Targeted delivery of drug conjugates to schwann cells and treatment methods in schwann cell-related diseases

Abstract

Disclosed are drug conjugates comprising a targeting moiety conjugated to a drug molecule, wherein the targeting moiety binds to a cell adhesion moiety and / or receptor expressed on a Schwann cell to mediate targeted delivery of the drug conjugate to the Schwann cell, and wherein the drug molecule modifies expression or activity of a disease-associated molecule, and / or confers a cytotoxic effect, in the Schwann cell. Further disclosed herein are methods of targeted delivery of a drug to the Schwann cells.

Description

TARGETED DELIVERY OF DRUG CONJUGATES TO SCHWANN CELLS ANDTREATMENT METHODS IN SCHWANN CELL-RELATED DISEASESBACKGROUND

[0001] Schwann cells, also called neurilemma cells, produce the myelin sheath around neuronal axons in the peripheral nervous system. They play a key role in the pathology of various inflammatory, metabolic, hereditary neuropathies and cancer (see, e.g., Lehmann and Hoke, CNS Neurol Disord Drug Targets. 2010 Dec, 9(6): 801-806; Kamil K, et al. Front. Neurol. 2019,10:87; Kresak, J.L, and Walsh, M., J Pediatr Genet. 2016, 5: 98, which are incorporated by reference herein in its entirety).

[0002] Some peripheral neuropathies are associated with abnormal protein or gene expression in Schwann cells, and suppression or regulation of such protein or gene expression in Schwann cells has been contemplated to treat those peripheral neuropathies. However, the drug delivery methods in the current practice, such as deliveries via nanoparticle and viral delivery, such as Adeno-associated virus (AAV) possess disadvantages including delivery mode, nonspecific targeting, reduced targeting efficiency, immunogenicity, generation of anti-drug antibodies (AD As) or safety concerns. As such, there exists a need to specifically target Schwann cells to manage, treat, or alleviate the large repertoire of Schwann cell-associated diseases.SUMMARY OF DISCLOSURE

[0003] Recognizing the need for a more effective and more targeted delivery of therapeutic compositions to Schwann cells, the present disclosure, in one aspect, discloses a drug conjugate comprising a targeting moiety conjugated to a drug molecule, wherein the targeting moiety binds to a cell adhesion moiety and / or receptor expressed on a Schwann cell to mediate targeted delivery of the drug conjugate to the Schwann cell, and wherein the drug molecule modifies expression or activity of a disease-associated molecule, and / or confers a cytotoxic effect, in the Schwann cell.

[0004] It is one embodiment, to provide a drug conjugate comprising a targeting moiety conjugated to a drug molecule, wherein the targeting moiety binds to a cell adhesion moiety and / or receptor expressed on a Schwann cell to mediate targeted delivery of the drug conjugate to the Schwann cell, and wherein the drug molecule modifies expression or activity of a disease- associated molecule, and / or confers a cytotoxic effect, in the Schwann cell.

[0005] It is a further embodiment, to provide the drug conjugate as described above, wherein the cell adhesion moiety and / or receptor is selected from a group consisting of Gliomedin, a leprosy receptor e.g., Laminin alpha 2-G4-5 (LNa2G), alpha-Dystroglycan (alpha-DG) and Myelin Protein zero (P0 protein), TAM (Tyro3, Axl, Mer) receptor, cell adhesion molecule (Cadm), or any Schwann cell specific receptor.

[0006] It is a further embodiment, to provide the drug conjugate as described in any of the above, wherein the cell adhesion moiety and / or receptor is selected from Nectin-like protein (Neel) e.g. Necll, Necl3, Necl2 and Necl4, SynCAM e.g. SynCAM4, Cadm e.g. Cadml- Cadm3, Neurofascinl55 (NF155), TAG1, myelin-associated glycoprotein (MAG), and neuronal cell adhesion molecule (NrCAM).

[0007] It is a further embodiment, to provide the drug conjugate as described in any of the above, wherein the targeting moiety is an antibody or antigen binding fragment thereof or a ligand molecule.

[0008] It is a further embodiment, to provide the drug conjugate as described in any of the above, wherein the targeting moiety is a ligand molecule, and wherein the ligand molecule comprises a protein, peptide, a glycoprotein, a glycan, a carbohydrate moiety, a fatty acid, a dendrimer, or a synthetic small molecule.

[0009] It is a further embodiment, to provide the drug conjugate as described in any of the above, wherein the targeting moiety is selected from anti-human Gliomedin Antibody, anti- Cadm4 mAb244 / 5 (NeuroMAB), Necl4-Fc antibody, anti-SynCAM4 Antibody, IGSF4C / SynCAM4 Antibody, neurofascin- 186 (NF 186) or a portion or derivative thereof, glycolipid PGL-1 or a portion or derivative thereof, trisaccharide of PGL-1 or a portion or derivative thereof, ML-LBP21 (histone-like protein / Hlp) or a portion or derivative thereof.

[0010] It is a further embodiment, to provide the drug conjugate as described in any of the above, wherein the antibody or antigen binding fragment thereof comprises IgG (Immunoglobulin G), including IgGl, IgG2a IgG2b, IgG3 and IgG4, IgA (Immunoglobulin A), including IgAl and IgA2, IgM (Immunoglobulin M), IgE (Immunoglobulin E), IgD (Immunoglobulin D), fragment antigen-binding (Fab fragment), fragment crystallizable region (Fc region), monovalent Fab’, divalent Fab2, single-chain variable fragment (scFv), diabody, minibody, nanobody, single-domain antibody (sdAb), (scFv)2, Fab, Fab', F(ab')2, Fv, dAb, Fd fragments, diabodies, F(ab')3, disulfide linked Fv, sdAb (VHH or nanobody), CDR (Complementarity-determining region), di-scFv, bi-scFv, tascFv (tandem scFv), triabody, tetrabody, V-NAR domain, Fcab, IgGACH2, DVD-Ig, probody, a DARPin, a Centyrin, anaffibody, an affilin, an affitin, an anticalin, an avimer, a Fynomer, a Kunitz domain peptide, a monoclonal antibody, a monobody (or adnectin), a tribody, and a nanofitin and mini-proteins or camelid antibody, or a monospecific, bispecific, trispecific, or a multi-specific antibody or binding fragment or antibody-mimetic thereof.

[0011] It is a further embodiment, to provide the drug conjugate as described in any of the above, wherein the antibody or antigen binding fragment thereof comprises binding specificity to Gliomedin protein.

[0012] It is a further embodiment, to provide the drug conjugate as described in any of the above, wherein the antibody or antigen binding fragment thereof comprises a complementary determining region (CDR) sequence having at least 90%, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity to a sequence selected from SEQ ID NO: 3-22 and 30 or a fragment, functional variant, or a combination thereof.

[0013] It is a further embodiment, to provide the drug conjugate as described in any of the above, wherein the antibody or antigen binding fragment thereof comprises a heavy chain variable region (VH) comprising heavy chain complementarity determining region (CDR) selected from CDR-H1, CDR-H2 and CDR-H3 or any combination thereof, and / or a light chain variable region (VL) comprising light chain CDR selected from CDR-L1, CDR-L2, and CDR- L3, or any combination thereof, and wherein the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR- L2, and CDR-L3, respectively, comprise a sequence having at least 90% identity to a sequence selected from:(a) the amino acid sequences of CDR-H1 comprising SEQ ID NO: 3, 4, 9 and 10;(b) the amino acid sequences of CDR-H2 comprising SEQ ID NO: 5, 6, 11 and 12;(c) the amino acid sequences of CDR-H3 comprising SEQ ID NO: 7, 8, 13 and 14;(d) the amino acid sequences of CDR-L1 comprising SEQ ID NO: 15, 16 and 20;(e) the amino acid sequences of CDR-L2 comprising SEQ ID NO: 17, 18, 21 and 30;(f) the amino acid sequences of CDR-L3 comprising SEQ ID NO: 19 and 22; or any functional variant, fragment or a combination thereof.

[0014] It is a further embodiment, to provide the drug conjugate as described in any of the above, wherein the drug molecule comprises an oligonucleotide, a peptide, a protein, an immunoglobulin, a small molecule, or a complex or any combination thereof.

[0015] It is a further embodiment, to provide the drug conjugate as described in any of the above, wherein the drug molecule comprises a degrader molecule (such as a protein degrader), a toxin such as Auristatin derivative, Maytansinoids, Calicheamicins, Pyrrolobenzodiazepines,Duocarmycins, Topoisomerase Inhibitors, a-Amanitin, Epothilones, cell death inducing molecules, cytotoxic agent, and / or any combination thereof.

[0016] It is a further embodiment, to provide the drug conjugate as described in any of the above, wherein the drug molecule comprises enzymes, enzyme inhibitors, proteins, kinase and / or phosphatase inhibitors or any protein-specific modulating agent.

[0017] It is a further embodiment, to provide the drug conjugate as described in any of the above, wherein the drug molecule comprises a double-stranded RNAi molecule or a singlestranded antisense oligonucleotide.

[0018] It is a further embodiment, to provide the drug conjugate as described in any of the above, wherein the drug molecule comprises RNA molecule such as tRNA, rRNA and mRNA, coding (cRNA), noncoding RNA (ncRNA), micro RNA (miRNA), small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), small-interfering RNA (siRNA), PlWI-interacting RNA (piRNA), short hairpin RNA (shRNA), microRNA (miRs), a double-stranded RNAi molecule, a single-stranded antisense oligonucleotide, antisense oligonucleotides (ASOs) and antiparallel triplex forming oligonucleotides.

[0019] It is a further embodiment, to provide the drug conjugate as described in any of the above, wherein the drug molecule comprises siRNA molecule comprising a nucleic acid sequence which has at least 95%, such as at least 96%, 97%, 98% or at least 99% sequence identity with a sequence selected from SEQ ID NO: 25-29, a complementary sequence thereof, or any combination thereof.

[0020] It is a further embodiment, to provide the drug conjugate as described in any of the above, wherein the drug molecule is conjugated with the targeting moiety via a linker.

[0021] It is a further embodiment, to provide the drug conjugate as described in any of the above, wherein the linker is a cleavable linker, a non-cleavable linker, covalently bound linker such as by chemical conjugation, linkers based on non-covalent interactions, a carbohydrate linker, a peptide linker, a glycan linker, or a non-peptide linker. Examples for such linkers include, but not limited to, Hydrazone Linkers, Disulfide Linkers, Peptide Linkers (Valinecitrulline (Val-Cit) and Alanine-alanine (Ala-Ala)), Beta-Glucuronide Linkers, Thioether Linkers (such as SMCC (succinimidyl-4-(N-maleimidomethyl)cyclohexane-l -carboxylate)), PEG Linkers, Conditional Linkers (such as Azobenzene Linkers and Diselenide Linkers).

[0022] It is a further embodiment, to provide the drug conjugate as described in any of the above, wherein the Drug molecule to the Targeting Moiety Ratio (DTMR) is in the range of 1 to 10 or characterized by a DTMR between 2 to 8, such as DTMR between 2 to 5.

[0023] It is a further embodiment, to provide the drug conjugate as described in any of the above, wherein the disease is selected from peripheral nervous system (PNS) disorders related to dysfunction and / or degeneration of Schwann cells and / or axons, and axon related disorders.

[0024] It is a further embodiment, to provide the drug conjugate as described in any of the above, wherein the disease is selected from peripheral demyelinating disease, peripheral neuropathy and / or hereditary motor and sensory neuropathy such as Charcot-Marie-Tooth disease (CMT), Guillain-Barre syndrome (acute inflammatory demyelinating polyradiculopathy type), chronic inflammatory demyelinating polyneuropathy, nerve trauma, neuropathic pain, post-chemotherapy neuropathy, diabetic neuropathy, diabetic peripheral neuropathy, migraine, fibromyalgia, demyelinating diseases such as multiple sclerosis, and malignant peripheral nerve sheath tumors (MPNSTs), endometriosis abdominopelvic pain and nerve injury.

[0025] It is a further embodiment, to provide the drug conjugate as described in any of the above, wherein the disease is Charcot-Marie-Tooth disease type 1 (CMT1), such as CMT type 1A (CMT1A).

[0026] It is a further embodiment, to provide the drug conjugate as described in any of the above, wherein the disease is a nerve tumor disease such as schwannomatosis, schwannomas and neurofibromatosis type 1 (NFl).

[0027] It is a further embodiment, to provide the drug conjugate as described in any of the above, wherein the disease-associated molecule is at least one of peripheral myelin protein 22 (PMP22), SMARCB1 encoded SWI / SNF subunit, LZTR1, chronic inflammatory demyelinating polyneuropathy (CIDP), Histone deacetylase-6 (HDAC6), P2X7 receptors, Neurotrophin-3 (NT-3), antiganglioside antibodies, enzymes, phosphatases, kinases, receptor tyrosine kinases (RTKs), Nonreceptor protein tyrosine kinases (nrPTKs), PMP2, MPZ, LITAF, FBLN5, EGR2, HDAC6, NEFL, GDAP1, MTMR2, SH3TC2, NDRG1, PRX, FGD4, SBF1, SBF2, FIG4, CTDP1, SURF1, ADCY6, CNTNAP1, HK1, MFN2, LRSAM1, NEFH, KIF5A, ATP1A1, VCP, TFG, DHTKD1, TUBB3, NAGLU, DCAF8, DGAT2, M0RC2, HSPB1, HSPB3, HSPB8, GARS, AARS, HARS, MARS, DYNC1H1, BICD2, REEP1, BSCL2, SETX, SLC5A7, MYH14, TRPV4, RAB7LMNA, PNKP, TRIM2, SPG11, MME, MCM3AP, SLC25A46, SCO2, MPV17, C12 or f65, IGHMBP2, SIGMAR1, VRK1, ATP7A, UBA1, GLE1, LAS1L, GJB1, YARS, INF2, DRP2, DNM2, GNB4, PDK3, GDAP1, COX6A1, PLEKHG5, KARS, AIFM1 and PRPS1.

[0028] It is a further embodiment, to provide a pharmaceutical composition comprising a drug conjugate as defined in any of the above and a pharmaceutically acceptable excipient.

[0029] It is a further embodiment, to provide the pharmaceutical composition as defined above, wherein the pharmaceutical composition is formulated for parenteral, systemic, intravenous, subcutaneous, or intrasciatic delivery.

[0030] It is a further embodiment, to provide a method of targeted delivery of a drug to a Schwann cell, comprising allowing contacting of the drug conjugate as defined in any of the above or the pharmaceutical composition as defined in any of the above to the Schwann cell, wherein the drug molecule comprises the drug.

[0031] It is a further embodiment, to provide the method as described in any of the above, wherein the drug is internalized into the Schwann cell upon binding of the targeting moiety to the cell adhesion moiety and / or receptor on the Schwann cell.

[0032] It is a further embodiment, to provide a method of treating or diagnosing a subject with a disease associated with dysfunction and / or degeneration of Schwann cells and / or axons, and axon related disorders, comprising administering to a subject in need of such a treatment or diagnosis an effective amount of the drug conjugate as defined in any of the above or a pharmaceutical composition as defined in any of the above.

[0033] It is a further embodiment, to provide the method as described in any of the above, wherein the disease is selected from schwannomatosis, schwannomas and neurofibromatosis type 1 (NF1) and the drug molecule is a degrader molecule (such as a protein degrader), a toxin such as Auristatin derivative, Maytansinoids, Calicheamicins, Pyrrolobenzodiazepines, Duocarmycins, Topoisomerase Inhibitors, a-Amanitin, Epothilones, cell death inducing molecules, cytotoxic agent, and / or any combination thereof.

[0034] It is a further embodiment, to provide the method as described in any of the above, wherein the disease is selected from peripheral demyelinating disease, peripheral neuropathy and / or hereditary motor and sensory neuropathy such as Charcot-Marie-Tooth disease (CMT), Guillain-Barre syndrome (acute inflammatory demyelinating polyradiculopathy type), chronic inflammatory demyelinating polyneuropathy, nerve trauma, neuropathic pain, postchemotherapy neuropathy, diabetic neuropathy, diabetic peripheral neuropathy, migraine, fibromyalgia, demyelinating diseases such as multiple sclerosis, and malignant peripheral nerve sheath tumors (MPNSTs), endometriosis abdominopelvic pain and nerve injury, and the drug molecule is an oligonucleotide, a peptide, a protein, an immunoglobulin.

[0035] It is a further embodiment, to provide the method as described in any of the above, wherein the drug molecule comprises siRNA molecule comprising a nucleic acid sequence which has at least 95%, such as at least 96%, 97%, 98% or at least 99% sequence identity with a sequence selected from SEQ ID NO: 25-29, a complementary sequence thereof, or any combination thereof.

[0036] It is a further embodiment, to provide an antibody or antigen binding fragment thereof having an anti-Gliomedin activity comprising a complementary determining region (CDR) sequence having at least 90%, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity to a sequence selected from SEQ ID NO: 3-22 and 30 or a fragment, functional variant or a combination thereof.

[0037] It is a further embodiment, to provide the antibody or antigen binding fragment thereof as described in any of the above, wherein the antibody or antigen binding fragment thereof comprises binding specificity to Gliomedin protein.

[0038] It is a further embodiment, to provide the antibody or antigen binding fragment thereof as described in any of the above, wherein the antibody or antigen binding fragment thereof comprises a heavy chain variable region (VH) comprising heavy chain complementarity determining region (CDR) selected from CDR-H1, CDR-H2 and CDR-H3 or any combination thereof, and / or a light chain variable region (VL) comprising light chain CDR selected from CDR-L1, CDR-L2, and CDR-L3, or any combination thereof, and wherein the CDR-H1, CDR- H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, respectively, comprise a sequence having at least 90%, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to a sequence selected from:(a) the amino acid sequences of CDR-H1 comprising SEQ ID NO: 3, 4, 9 and 10;(b) the amino acid sequences of CDR-H2 comprising SEQ ID NO: 5, 6, 11 and 12;(c) the amino acid sequences of CDR-H3 comprising SEQ ID NO: 7, 8, 13 and 14;(d) the amino acid sequences of CDR-L1 comprising SEQ ID NO: 15, 16 and 20;(e) the amino acid sequences of CDR-L2 comprising SEQ ID NO: 17, 18, 21 and 30;(f) the amino acid sequences of CDR-L3 comprising SEQ ID NO: 19 and 22; or any fragment or a combination thereof.

[0039] It is a further embodiment, to provide the antibody or antigen binding fragment thereof as described in any of the above, wherein the antibody or antigen binding fragment thereof comprises an amino acid sequence which has at least 90%, such as at least 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or at least 99% identity with SEQ ID NO: 1, SEQ ID NO:2 or a combination thereof.

[0040] It is a further embodiment, to provide a use of an amino acid sequence comprising a complementary determining region (CDR) sequence having at least 90%, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity to a sequence selected from SEQ ID NO: 3-22 and 30 or a fragment, functional variant or a combination thereof, as an anti-Gliomedin antibody or antigen binding fragment thereof.

[0041] It is a further embodiment, to provide the use as described in any of the above, wherein the antibody or antigen binding fragment thereof comprises binding specificity to Gliomedin protein.

[0042] It is a further embodiment, to provide the use as described in any of the above, wherein the antibody or antigen binding fragment thereof comprises a heavy chain variable region (VH) comprising heavy chain complementarity determining region (CDR) selected from CDR-H1, CDR-H2 and CDR-H3 or any combination thereof, and / or a light chain variable region (VL) comprising light chain CDR selected from CDR-L1, CDR-L2, and CDR-L3, or any combination thereof, and wherein the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, respectively, comprise a sequence having at least 90%, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to a sequence selected from:(a) the amino acid sequences of CDR-H1 comprising SEQ ID NO: 3, 4, 9 and 10;(b) the amino acid sequences of CDR-H2 comprising SEQ ID NO: 5, 6, 11 and 12;(c) the amino acid sequences of CDR-H3 comprising SEQ ID NO: 7, 8, 13 and 14;(d) the amino acid sequences of CDR-L1 comprising SEQ ID NO: 15, 16 and 20;(e) the amino acid sequences of CDR-L2 comprising SEQ ID NO: 17, 18, 21 and 30;(f) the amino acid sequences of CDR-L3 comprising SEQ ID NO: 19 and 22; or any fragment or a combination thereof.

[0043] It is a further embodiment, to provide the use as described in any of the above, wherein the antibody or antigen binding fragment thereof comprises at least 90%, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity with SEQ ID NO: 1, SEQ ID NO:2 or a combination thereof, as an anti-Gliomedin antibody or antigen binding fragment thereof.

[0044] It is a further embodiment, to provide a nucleic acid encoding the antibody or antigen binding fragment thereof as defined in any of the above.

[0045] It is a further embodiment, to provide a host cell comprising the nucleic acid as defined above.

[0046] It is a further embodiment, to provide a method for producing the antibody or antigen binding fragment thereof as defined in any of the above, which comprises the steps of culturing the host cell as defined above, and recovering the polypeptides from the cell culture.

[0047] It is a further embodiment, to provide an isolated polynucleotide having siRNA activity for reducing human PMP22 expression, comprising a nucleic acid sequence which has at least 95%, such as at least 96%, 97%, 98% or at least 99% identity with SEQ ID NO:25-29 or a combination thereof.

[0048] It is a further embodiment, to provide a use of a nucleic acid sequence comprising at least 95%, such as at least 96%, 97%, 98% or at least 99% identity with SEQ ID NO:25-29 or a combination thereof, as siRNA molecule for reducing PMP22 expression in a Schwann cell.

[0049] It is a further embodiment, to provide a composition comprising the antibody or antigen binding fragment thereof as defined in any of the above and / or the isolated polynucleotide as defined in any of the above and a pharmaceutically acceptable carrier.

[0050] It is a further embodiment, to provide a drug conjugate comprising a targeting moiety conjugated to a drug molecule, wherein the targeting moiety binds to a cell adhesion moiety and / or receptor expressed on a Schwann cell to mediate targeted delivery of the drug conjugate to the Schwann cell, and the drug molecule modifies expression or activity of a disease- associated molecule, and / or confers a cytotoxic effect, in the Schwann cell, wherein the targeting moiety comprises the antibody or antigen-binding fragment as defined in any of the above.INCORPORATION BY REFERENCE

[0051] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.BRIEF DESCRIPTION OF THE DRAWINGS

[0052] The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings (also “Figure”, "Fig." and “FIG.” herein), of which:

[0053] FIG. 1A is a photographic illustration of SDS-PAGE (SDS-Polyacrylamide Gel Electrophoresis) analysis of mouse anti-Gliomedin antibody (Ab) purification. The lanes from left to right represent separation of molecular weight markers (M), reduced form (R3) and nonreduced form (N3) of the mouse anti-Gliomedin antibody;

[0054] FIG. IB is a graphic illustration of SEC (Size Exclusion Chromatography) analysis (Superdex200, 60ml, XK16 / 30) of mouse anti-Gliomedin Ab purification;

[0055] FIG. 2 is a graphic illustration of ELISA (enzyme-linked immunosorbent assay) binding mouse anti-Gliomedin Ab against human Gliomedin-ECD-Fc recombinant protein;

[0056] FIG. 3A is a graphic illustration of FACS (Fluorescence-activated cell sorting) analysis using the primary antibody mouse anti-Gliomedin- 1 (internal synthesis, Lot: #CP2024090301) lOOnM, validating the expression of human Gliomedin (hGLDN) in CHOK1 cells infected by hGLDN and PMP22 lentivirus;

[0057] FIG. 3B is a graphic illustration of FACS (Fluorescence-activated cell sorting) analysis using the primary antibodies Rabbit anti-PMP22 antibody validating the stable expression of human PMP22 in CHOK1 cells infected by hGLDN and PMP22 lentivirus; and

[0058] FIG. 4 is a graphic illustration of reduction of hPMP22 expression induced by different siRNA variants against hPMP22 gene evaluated by the transduction of CHO- transfected hPMP22 / hGliomedin cells as compared to control non-treated cells.DETAILED DESCRIPTION

[0059] Cancer is the second leading cause of human death next to coronary disease. Worldwide, millions of people die from cancer every year. In the United States alone, as reported by the American Cancer Society, cancer causes the death of well over a half-million people annually, with over 1.2 million new cases diagnosed per year.

[0060] Recognizing the need for a more effective and more targeted delivery of therapeutic compositions to Schwann cells and particularly to and for Schwann cell-related oncologic indications, the present disclosure, in one aspect, provides a drug conjugate comprising atargeting moiety conjugated to a drug molecule, wherein the targeting moiety binds to a receptor, or a cell adhesion moiety expressed on a Schwann cell to mediate targeted delivery of the drug conjugate to the Schwann cell, and wherein the drug molecule modifies expression or activity of a disease-associated molecule, and / or confers cytotoxic effect or activity, in the Schwann cell.

[0061] It is noted that according to embodiments of the present invention, the use of a drug molecule inducing a cytotoxic activity or conferring a cytotoxic effect in the Schwann cells (e.g. a cytotoxic agent) is mainly targeted or directed for the treatment of cancer-related or associated disorders such as Schwann cell-related oncologic indications, e.g. schwannoma or schwannomatosis.

[0062] According to further aspects, the present invention provides a drug conjugate that is an oligonucleotide conjugate, e.g. antibody-oligonucleotide conjugate (AOC). In this embodiment the drug molecule is an oligonucleotide, such as small interfering RNA (siRNA). Such a drug conjugate is useful for treating neuroscience, neuromuscular and CNS indications e.g., neuromuscular disorder Charcot-Marie-Tooth (CMT) disease, such as CMT type 1 A (CMT1 A), axonal loss, dysfunction of the neuronal axon, or abnormal axon-Schwann cell interaction. For example, it decreases the expression of PMP22 protein (CMT associate molecule) in Schwann cells, which is the target cell type for the development, maintenance, and function of peripheral nerves. The oligonucleotide conjugate facilitates the targeted approach of the present invention by conjugating RNA interference (RNAi) variants to targeting moieties, thereby enhancing targeted distribution and cellular uptake to specific tissues and cell types of interest, and modulating gene expression for specific disease-associated molecules.

[0063] Reference is now made to Charcot-Marie-Tooth (CMT) disease which collects a genetically heterogeneous group of inherited disorders sharing a common core phenotype of length-dependent progressive sensory-motor or motor neuropathy with foot deformities and altered deep tendon reflexes. It is classified according to nerve conduction studies into a demyelinating and an axonal variety: CMT1 for demyelinating (with slowed nerve conduction velocities, NCVs) neuropathies with autosomal dominant inheritance, CMT4 for autosomal recessive demyelinating forms, and CMT2 for primary axonal types with autosomal dominant or recessive transmission. There are intermediate forms of CMT with NCV values falling in between CMT1 and CMT2: the most relevant is CMTX1, which is associated with mutations in the GJB1 gene on chromosome Xql3.1; less common autosomal dominant or recessive forms are on the record. Pure motor forms of CMT are characterized by the preservation ofsensory nerves and are labelled distal Hereditary Motor Neuropathies (dHMNs), which can have autosomal dominant, autosomal recessive, or X-linked transmission. Pure or predominantly sensory neuropathies are grouped under the term of Hereditary Sensory (and Autonomic) Neuropathies (HS(A)Ns) and can have autosomal dominant or recessive inheritance patterns. Eventually, Hereditary Neuropathy with liability to Pressure Palsies is an autosomal dominant disorder characterized by recurrent focal neuropathies, which are typically painless and transient, and it is associated with the deletion of the PMP22 gene, whereas the duplication of the same gene with PMP22 overexpression is associated with the most frequent CMT subtype, CMT1 A. Overall, this group of inherited peripheral neuropathies is also labelled as CMT and related neuropathies and is further classified according to the mutated gene. Indeed, about 100 genes have been associated with CMT and related disorders, which code for proteins involved in many different functions relevant for the nerves, including myelin formation and maintenance, axonal transport, vesicular trafficking, mitochondrial homeostasis, Endoplasmic Reticulum (ER) stress, channels formation, etc. (see Chiara Pisciotta, Paola Saveri & Davide Pareyson (2021) Updated review of therapeutic strategies for Charcot-Marie-Tooth disease and related neuropathies, Expert Review of Neurotherapeutics, 21:6, 701-713, DOI: 10.1080 / 14737175.2021.1935242; Pisciotta, C.; Saveri, P.; Pareyson, D. Challenges in Treating Charcot-Marie-Tooth Disease and Related Neuropathies: Current Management and Future Perspectives. Brain Sci. 2021, 11, 1447. doi.org / 10.3390 / brainscil l 111447).

[0064] Antibodies have a high-degree of specificity and identification abilities with respect to their corresponding antigens or cell-surface receptors. On the other hand, many cytotoxic drug molecules cannot be used for cancer therapy because they cannot selectively kill cancer cells. Therefore, the linking or contacting of antibodies and highly toxic drugs (such as toxins) generates a highly selective and specific conjugated drug to be delivered for specific target cells or receptor molecules. It is further within the scope of the present invention that conjugating antibodies to oligonucleotides, such as RNAi variants, enhances the targeted delivery of the RNAi variant to the disease-associated molecule of interest, thereby modulating the expression of the gene of interest.

[0065] It is within the scope of the present invention that a drug conjugate such as Antibody- Drug Conjugate (ADC) comprises targeting moieties (e.g. antibodies), optionally linkers, and drugs (such as toxins). However, these toxins appear to be highly cytotoxic (IC5CX1 nM) due to the high dose required for their performance / efficacy.

[0066] In some instances, the cell adhesion moiety or receptor as disclosed herein is selected from a group consisting of Gliomedin, a leprosy receptor (e.g., Laminin alpha 2-G4-5 (LNa2G), P0 protein and alpha-Dystroglycan (alpha-DG)), TAM (Tyro3, Axl, Mer) receptor, cell adhesion molecule (Cadm), or any Schwann cell specific receptor. In some instances, the cell adhesion moiety or receptor is Gliomedin. In some instances, the cell adhesion moiety or receptor is Cadm. In some instances, the cell adhesion moiety or receptor is a leprosy e.g., Laminin alpha 2-G4-5 (LNa2G), alpha-Dystroglycan (alpha-DG) and Myelin Protein zero (P0 protein). In some instances, the cell adhesion moiety or receptor is Tyro3, Axl, Mer (TAM) receptor. In some instances, the cell adhesion moiety and / or receptor is selected from Nectin- like protein (Neel) e.g. Necll, Necl3, Necl2 and Necl4, SynCAM e.g. SynCAM4, Cadm e.g. Cadml-Cadm3, Neurofascinl55 (NF155), TAG1, myelin-associated glycoprotein (MAG), and neuronal cell adhesion molecule (NrCAM).In some instances, the targeting moiety is an antibody or antigen, a binding fragment thereof or a ligand molecule. In some instances, the antibody is a fully human antibody, and is recombinantly produced. The antibody or antigen binding fragment thereof may comprise IgG (Immunoglobulin G), including IgGl, IgG2a IgG2b, IgG3 and IgG4, IgA (Immunoglobulin A), including IgAl and IgA2, IgM (Immunoglobulin M), IgE (Immunoglobulin E), IgD (Immunoglobulin D), fragment antigenbinding (Fab fragment), fragment crystallizable region (Fc region), monovalent Fab’, divalent Fab2, single-chain variable fragment (scFv), diabody, minibody, nanobody, single-domain antibody (sdAb), (scFv)2, Fab, Fab', F(ab')2, Fv, dAb, Fd fragments, diabodies, F(ab')3, disulfide linked Fv, sdAb (VHH or nanobody), CDR (Complementarity-determining region), di-scFv, bi-scFv, tascFv (tandem scFv), triabody, tetrabody, V-NAR domain, Fcab, IgGACH2, DVD-Ig, probody, a DARPin, a Centyrin, an affibody, an affilin, an affitin, an anticalin, an avimer, a Fynomer, a Kunitz domain peptide, a monoclonal antibody, a monobody (or adnectin), a tribody, and a nanofitin and mini-proteins or camelid antibody or a monospecific, bispecific, trispecific, or a multi-specific antibody or binding fragment or antibody-mimetic thereof or a bispecific antibody, or a multi-specific antibody or binding fragment or antibodymimetic thereof. In some instances, the targeting moiety is a ligand molecule, and wherein the ligand molecule comprises a protein, a peptide, a glycoprotein, a glycan, a carbohydrate moiety, a fatty acid, a dendrimer, or a synthetic small molecule.

[0067] In some instances, the targeting moiety is any antigen-binding variant that comprises a CDR sequence having at least 90% sequence identity, such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% toa sequence selected from SEQ ID NO: 3-22, and 30 or any fragment or functional variant thereof.

[0068] In some instances, the drug molecule comprises a peptide or a small molecule, such as an oligonucleotide and / or toxin.

[0069] In some instances, the drug molecule comprises toxins commonly or conventionally used, as an example, Auristatin derivative such as Monomethyl auristatin E (MMAE) and Monomethylauristatin F (MMAF).

[0070] In some instances, the drug molecule comprises maytansinoids, calicheamicins, pyrrolobenzodiazepines, duocarmycins, topoisomerase inhibitors, a-Amanitin, epothilones, cell death inducing molecules, cytotoxic agent, and / or any combination thereof.

[0071] It is within the scope of the present invention that the mentioned above toxins, cell death inducing molecules and cytotoxic agents are herein as drug molecules within the drug conjugate for treatment of a nerve tumor disease such as schwannomatosis, schwannomas and neurofibromatosis type 1 (NF1).

[0072] In some embodiments, the drug molecule comprises a double-stranded RNAi molecule, a single-stranded antisense oligonucleotide or a protein degrader molecule.

[0073] In some instances, the drug molecule is conjugated with the targeting moiety via a linker. In some instances, the linker is a cleavable linker or a non-cleavable linker.

[0074] In some instances, the Drug molecule to the Targeting Moiety Ratio (DTMR) is about 1 to 10.

[0075] In some embodiments, the Drug-Targeting Moiety Ratio (DTMR), as measured according to Matsuda, Y., Mendelsohn, BA., Chem Pharm Bull (Tokyo). 2021 ;69, 976 is between 2 to 8. In some instances, the DTMR can potentially range from 0 to 8. In some instances, an average DTMR is between 3 to 4.

[0076] It is herein acknowledged that therapeutic targeted drug conjugates as disclosed herein have different DTMRs. The specific DTMR may vary depending on several factors, including:

[0077] - The type of the targeting moiety (e.g. antibody): Different targeting moieties (e.g. antibodies) have different structures and properties that can affect the amount of drug molecules that can be attached.

[0078] - The type of drug molecule: Different drugs have different sizes and potencies, which can also affect the DTMR.

[0079] - The desired therapeutic effect: The aim is to find a balance between delivering sufficient drug to kill cancer cells or to modify expression or activity of a disease associatedmolecule, while minimizing side effects. Thus, the drug conjugate of the present invention is aimed to achieve the maximal therapeutic efficacy with minimum drug dosage.

[0080] Examples of drug conjugates (e.g. ADCs) within the scope of the present invention and their corresponding DTMRs are presented in Table 1.Table 1: DTMR values of different ADCs

[0081] In some aspects, provided herein is a pharmaceutical composition comprising the drug conjugate described herein and a pharmaceutically acceptable excipient. In some instances, the pharmaceutical composition is formulated for parenteral, intravenous, systemic, subcutaneous, intrathecal, or intrasciatic delivery.

[0082] In some aspects, provided herein is a method of targeted delivery of a drug to a Schwann cell, comprising contacting the drug conjugate described herein or the pharmaceutical composition described herein to the Schwann cell, wherein the drug molecule comprises the drug or a precursor of the drug. In some instances, the drug or the precursor of the drug is internalized into the Schwann cell upon binding of the targeting moiety to the receptor on the Schwann cell and exerts biological activities of the drug or the precursor of the drug.

[0083] Provided herein are drug conjugates comprising targeting moieties conjugated to drug molecules, wherein the targeting moieties bind to receptors expressed on Schwann cells to mediate targeted delivery of the drug conjugate to the Schwann cell, and wherein the drug molecules modify expression or activity of disease-associated molecules in the Schwann cells. Further disclosed herein are methods of targeted delivery of a drug to the Schwann cells. Alsodisclosed herein are methods of delivering of a drug to Schwann cell by conjugating the drug to a targeting moiety that specifically or preferentially target the Schwann cell. Also disclosed herein are methods of treating or preventing a peripheral demyelinating disease or a peripheral neuropathy, or alleviating or reducing symptoms of the peripheral demyelinating disease or the peripheral neuropathy in a subject in need thereof.

[0084] As used herein the term "about" denotes ± 25% of the defined amount or measure or value.

[0085] As used herein the term "similar" denotes a correspondence or resemblance range of about ± 20%, particularly ± 15%, more particularly about ± 10% and even more particularly about ± 5%.

[0086] As used herein the term "corresponding" generally means similar, analogous, like, alike, akin, parallel, identical, resembling or comparable. In further aspects it means having or participating in the same relationship (such as type or species, kind, degree, position, correspondence, or function). It further means related or accompanying. In some embodiments of the present invention, it refers to related sequences (nucleic acid or polypeptide).

[0087] As used herein, the terms "nucleic acid", "nucleic acid sequence", "nucleotide", "nucleic acid molecule" or "polynucleotide" are intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), natural occurring, mutated, synthetic DNA or RNA molecules, and analogs of the DNA or RNA generated using nucleotide analogs. It can be single-stranded or double-stranded. Such nucleic acids or polynucleotides include, but are not limited to, coding sequences of structural genes, anti-sense sequences, and non-coding regulatory sequences that do not encode mRNAs or protein products. These terms also encompass a gene. The term "gene", "allele" or "gene sequence" is used broadly to refer to a DNA nucleic acid associated with a biological function. Thus, genes may include introns and exons as in the genomic sequence, or may comprise only a coding sequence as in cDNAs, and / or may include cDNAs in combination with regulatory sequences. Thus, according to the various aspects of the invention, genomic DNA, cDNA or coding DNA may be used. In one embodiment, the nucleic acid is cDNA or coding DNA.

[0088] The terms "peptide", "polypeptide" and "protein" are used interchangeably herein and refer to amino acids, including non-natural amino acids, in a polymeric form of any length, linked together by peptide bonds.

[0089] As used herein the term "average" refers to the mean value as obtained by measuring a predetermined parameter in each individual of a certain population and calculating the mean value according to the number of individuals in said population.

[0090] The term "consists essentially of' (and grammatical variants thereof), as applied to a polynucleotide or polypeptide sequence of this invention, means a polynucleotide or polypeptide sequence that consists of both the recited sequence (e.g., SEQ ID NO) and a total of ten or less (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional nucleotides or amino acid on the 5' and / or 3' ends of the recited sequence such that the function of the polynucleotide or polypeptide is not materially altered. The total of ten or less additional nucleotides or amino acids includes the total number of additional nucleotides or amino acids on both ends added together.

[0091] In the context of the present invention, the terms "homolog" and "homologous sequence" are often used to describe genetic sequences that share a common ancestry and exhibit a certain degree of identity or similarity. The term "homolog" refers to a polynucleotide or polypeptide sequence that shares a common evolutionary origin with another sequence (e.g. gene or protein or any fragment thereof). Homologs can be found within the same species (paralogs) or in different species (orthologs). They typically perform similar functions but may have diverged over time due to evolutionary pressures. The term "homologous sequence" as used herein refers to a DNA, RNA, or protein (polypeptide) sequence that is similar to another sequence. The degree of similarity can vary, and it is often quantified by sequence alignment techniques. Homologous sequences may indicate functional similarities and potential applications in biotechnology. The term “homology” further means polynucleotide or polypeptide sequences that are similar. For example, a “region of homology to a genomic region” is a region of DNA that has a similar sequence to a given “genomic region” in the cell or organism genome. A region of homology can be of any length that is sufficient to promote homologous recombination at the cleaved target site, such that the region of homology has sufficient homology to undergo homologous recombination or complementation with the corresponding genomic region. “Sufficient homology” indicates that two polynucleotide or polypeptide sequences have sufficient structural similarity to act as substrates for a homologous recombination reaction or have the same function. The structural similarity includes overall length of each polynucleotide or polypeptide fragment, as well as the sequence similarity of the polynucleotides or polypeptides.

[0092] Defining homologous sequences can be crucial for establishing the scope of the invention, as it may determine the range of genetic variations covered. This can include specifying a percentage of sequence identity or similarity that qualifies as homologous.

[0093] In the context of the embodiments of the invention, the term “sequence identity” refers, without limitation, to the occurrence of exactly the same or having a specified percentage of nucleotide or amino acid in the same position in aligned sequences. According to the present invention, the percent of identity or homology between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of identity percent between two sequences can be accomplished using a mathematical algorithm as known in the relevant art. As used herein, the term “sequence identity” is interchangeable with “sequence homology” or "sequence similarity".

[0094] It is further within the scope that the terms "similarity" and "identity" additionally refer to local homology, identifying domains that are homologous or similar (in nucleotide and / or amino acid sequence). It is acknowledged that bioinformatics tools such as BLAST, S SEARCH, FAS TA, and HMMER calculate local sequence alignments which identify the most similar region between two sequences. For domains that are found in different sequence contexts in different proteins, the alignment should be limited to the homologous domain, since the domain homology is providing the sequence similarity captured in the score. According to some aspects the term similarity or identity further includes a sequence motif, which is a nucleotide or amino-acid sequence pattern that is widespread and has, or is conjectured to have, a biological significance. Proteins may have a sequence motif and / or a structural motif, a motif formed by the three- dimensional arrangement of amino acids which may not be adjacent.

[0095] It is within the scope of the present invention, that homologous qualifies a percentage of at least about 90% identity or similarity, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity or similarity to a defined polypeptide sequence. It is further within the scope of the present invention that homologous qualifies a percentage of at least about 95% identity or similarity, such as at least 96%, 97%, 98%, or 99% identity or similarity to a defined polynucleotide sequence.

[0096] "Sequence similarity" can be described by the percent sequence identity over the whole length of the sequences, and / or by conserved regions comprising localized similarities such ascontiguous nucleotides or amino acids having 100% sequence identity, and percent sequence identity over a portion of the length of the sequences.

[0097] The amount of sequence identity shared by a target and a donor polynucleotide or polypeptide can vary and includes total lengths and / or regions having unit integral values in the ranges of about 1-20, 20-50, 50-100, 75-150, 100-250, 150-300, 200-400, 250- 500, 300-600, 350-750, 400-800, 450-900, 500-1000, 600-1250, 700- 1500, 800-1750, 900-2000, l-2.5k, 1.5- 3 k, 2-4 k, 2.5-5 k, 3-6 k, 3.5-7 k, 4-8 k, 5-10 k base pairs or amino acids, or up to and including the total length of the target site. These ranges include every integer within the range. The amount of homology can also be described by percent sequence identity over the full aligned length of the two polynucleotides or polypeptides which includes percent sequence identity (or similarity) of about at least 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. Sufficient homology includes any combination of polynucleotide or polypeptide length, global percent sequence identity, and optionally conserved regions of contiguous nucleotides or amino acids or local percent sequence identity. Sufficient homology can also be described by the predicted ability of two polynucleotides or polypeptides to specifically hybridize under high stringency conditions, see, for example, Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, NY); Current Protocols in Molecular Biology, Ausubel et al., Eds (1994) Current Protocols, (Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.); and, Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology- Hybridization with Nucleic Acid Probes, (Elsevier, New York).

[0098] The structural similarity between a given sequence region and the corresponding region of homology can be of any degree of sequence identity that allows for homologous recombination or hybridization, binding or recognition to occur. For example, the amount of homology or sequence identity shared by two sequences can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, such that the sequences undergo homologous recombination, hybridization or recognition or binding.

[0099] As used herein, "sequence identity" or "identity" in the context of two nucleic acid or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. When percentage of sequence identity is used in reference to proteins, it is recognized that residuepositions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. The term further refers hereafter to the amount of characters which match exactly between two different sequences. Hereby, gaps are not counted, and the measurement is relational to the shorter of the two sequences.

[0100] The term "functional variant" or "functional variant of a nucleic acid or amino acid sequence" as used herein refers to a sequence or part or fragment of a sequence which retains the biological function of the full non-variant and hence has the activity of the expressed gene or polypeptide. A functional variant also comprises a variant of a nucleic acid sequence of interest encoding a polypeptide which has sequence alterations that do not affect function of the resulting polypeptide, for example, in non-conserved residues or by degenerate, or redundant sequence encoding the same amino acids. Also encompassed is a variant that is substantially identical, i.e. has only some sequence variations, for example, in non-conserved residues and is biologically active.

[0101] Reference is now made to the term "antibody humanization" or " humanization" or "humanized antibody" which refers to a biotechnological process used to modify non- human antibodies, typically derived from animals like mice, to make them more compatible with the human immune system. This technique is crucial for developing therapeutic antibodies that are less likely to trigger immune responses, such as the production of anti-drug antibodies (AD As), which can neutralize the drug’s effects and cause adverse reactions. The process involves replacing the non-human constant and framework regions of the antibody with human sequences while preserving the antigen-binding complementarity-determining regions (CDRs), which maintain the antibody’s specificity. The humanization process includes sequencing the non-human antibody and selecting a human framework with structural similarity and low immunogenic potential. The CDRs are then grafted onto this framework, followed by computational and experimental optimization to enhance binding affinity, stability, and expression levels. Advanced techniques, such as deimmunization and structure-based design, further reduce immunogenicity and improve functionality. Humanized antibodies have become essential in treating various diseases, including cancers, autoimmune disorders, and infectious diseases, as they combine high specificity and therapeutic efficacy with minimized risk of immune rejection. Humaniztion of antibodies are described for example in WO1991009967A1.ConjugatesTargeting Moieties

[0102] Historically, the treatment of peripheral nervous system (PNS) disorders, including oncology related disorders has focused on non-targeted therapeutics or surgery, but the treatment has been shown with poor clinical outcomes. For certain PNS disorders the PNS disorders that are directly or indirectly related to the dysfunction of the Schwann cells, specific or preferred targeting of drugs to the Schwann cell can increase the efficacy and outcome of the treatment.

[0103] However, the specificity of the manipulation and / or modification of Schwann cells is a main roadblock to advance Schwann cell therapies. To meet such a need, provided herein in some instances is the target moiety that specifically directs cargoes or paylaoads to Schwann cells.

[0104] In some aspects, the targeting moiety disclosed herein binds to a receptor or a cell adhesion moiety expressed on a Schwann cell. In some aspects, the targeting moiety disclosed herein binds to a portion of cell adhesion moiety or receptor complex expressed on a Schwann cell. In some instances, the receptor or a cell adhesion moiety is a transmembrane molecule comprising an extracellular domain, and the targeting moiety binds to the extracellular domain of the cell adhesion moiety or receptor. In some instances, the cell adhesion moiety or receptor complex comprises an extracellular molecule that forms a complex with the cell adhesion moiety or receptor molecule (e.g., membrane bound cell adhesion moiety or receptor via transmembrane domain) directly or indirectly. In some instances, the targeting moiety binds to the extracellular molecule of the complex. In some instances, the targeting moiety binds to a cell adhesion molecule that mediates the interaction between myelinating Schwann cells and the axons they ensheath. In some instances, the cell adhesion moiety or receptor is expressed in the myelinated Schwann cells. In some instances, the cell adhesion moiety or receptor is highly expressed at the edge of myelin cells. In some instances, the cell adhesion moiety or receptor mediates the interaction of axon and glial cells or mediates axon-glial contact. In some instances, the cell adhesion moiety or receptor mediates the pathogen (or a portion thereof) uptake to the Schwan cells in certain disease conditions. As such, as used herein, the targeting moiety binding to a cell adhesion moiety or receptor also includes a targeting moiety that binds to a portion of a cell adhesion moiety or receptor complex that are naturally present in healthy or pathological conditions of Schwann cells.

[0105] In some embodiments, the targeting moiety is an antibody or antigen binding fragment thereof or a ligand molecule. In certain aspects, the antibody or antigen binding fragment thereof comprises IgG (Immunoglobulin G), including IgGl, IgG2a IgG2b, IgG3 and IgG4 isotypes, IgA (Immunoglobulin A), including IgAl and IgA2 isotypes, IgM (Immunoglobulin M), IgE (Immunoglobulin E), IgD (Immunoglobulin D), fragment antigen-binding (Fab fragment), fragment crystallizable region (Fc region), monovalent Fab’, divalent Fab2, singlechain variable fragment (scFv), diabody, minibody, nanobody, single-domain antibody (sdAb), (scFv)2, Fab, Fab', F(ab')2, Fv, dAb, Fd fragments, diabodies, F(ab')3, disulfide linked Fv, sdAb (VHH or nanobody), CDR (Complementarity-determining region), di-scFv, bi-scFv, tascFv (tandem scFv), triabody, tetrabody, V-NAR domain, Fcab, IgGACH2, DVD-Ig, probody, a DARPin, a Centyrin, an affibody, an affilin, an affitin, an anticalin, an avimer, a Fynomer, a Kunitz domain peptide, a monoclonal antibody, a monobody (or adnectin), a tribody, and a nanofitin and mini-proteins or camelid antibody, or a monospecific, bispecific, trispecific, or a multi-specific antibody or binding fragment or antibody-mimetic thereof. In some instances, the targeting moiety is a ligand molecule comprising a protein, a peptide, a glycoprotein, a glycan, a carbohydrate moiety, a fatty acid, a dendrimer, or a synthetic small molecule. In certain instances, the targeting moiety is selected from anti-Gliomedin antibody, anti-Cadm4 mAb244 / 5 (NeuroMAB), Necl4-Fc antibody, anti-SynC AM4 Antibody, IGSF4C / SynCAM4 Antibody, neurofascin-186 (NF 186) or a portion or derivative thereof, glycolipid PGL-1 or a portion or derivative thereof, trisaccharide of PGL-1 or a portion or derivative thereof, ML- LBP21 (histone-like protein / Hlp) or a portion or derivative thereof.

[0106] In some instances, the targeting moiety disclosed herein binds to a Gliomedin. In some instances, the targeting moiety disclosed herein binds to the extracellular region of a Gliomedin. In some instances, the targeting moiety disclosed herein binds to the C-terminal region of a gliomedin.

[0107] In some instances, the targeting moiety disclosed herein binds to a portion of the C- terminal region of a gliomedin. In some instances, the targeting moiety disclosed herein binds to the coiled-coil domain of a gliomedin. In some instances, the targeting moiety disclosed herein binds to the interrupted collagen repeats domain of a gliomedin. In some instances, the targeting moiety disclosed herein binds to the olfactomedin domain of a gliomedin. In some instances, the targeting moiety disclosed herein binds to amino acid residues 276-287 of rat Gliomedin NP 852047.2 (CVIPNDDTLVGRA). In some instances, the targeting moiety disclosed herein binds to amino acid residues 365-460 of a Gliomedin.

[0108] In some instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof which binds to a Gliomedin. In some instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof which binds to the extracellular region of a gliomedin. In some instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof which binds to the C-terminal region of a gliomedin. In some instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof which binds to a portion of the C-terminal region of a gliomedin. In some instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof which binds to the coiled-coil domain of a gliomedin. In some instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof which binds to the interrupted collagen repeats domain of a gliomedin. In some instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof which binds to the olfactomedin domain of a gliomedin. In some instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof which binds to amino acid residues 276-287 of rat Gliomedin NP 852047.2 (CVIPNDDTLVGRA). In some instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof which binds to amino acid residues 365-460 of a gliomedin.

[0109] In some instances, the targeting moiety disclosed herein is a commercially available antibody that binds to gliomedin. In some instances, the targeting moiety disclosed herein is anti-Gliomedin antibody (mAb94) disclosed in Eshed etal., Neuron, volume 47, issue 2, p215- 229, which is incorporated by reference in its entirety. In some instances, the targeting moiety disclosed herein is anti-GLDN antibody NovoPro #: 175453. In some instances, the targeting moiety disclosed herein is anti-GLDN antibody Thermo Fisher #:BS-11032R.

[0110] In some instances, the targeting moiety disclosed herein binds to a leprosy receptor. In specific instances, the targeting moiety disclosed herein binds to Laminin alpha 2-G4-5 (LNa2G) also referred to as laminin 2. In specific instances, the targeting moiety disclosed herein binds to the C-terminal region of laminin 2. In some instances, the targeting moiety disclosed herein binds to the a2-subunit of laminin 2. In some instances, the targeting moiety disclosed herein binds to G domain of the a2-subunit of laminin 2. In some instances, the targeting moiety disclosed herein binds to G1 domain of the a2-subunit of laminin 2. In some instances, the targeting moiety disclosed herein binds to G2 domain of the a2-subunit of laminin 2. In some instances, the targeting moiety disclosed herein binds to G3 domain of the a2- subunit of laminin 2. In some instances, the targeting moiety disclosed herein binds to G4domain of the a2-subunit of laminin 2. In some instances, the targeting moiety disclosed herein binds to G5 domain of the a2-subunit of laminin 2. In some instances, the targeting moiety disclosed herein binds to the 01 -subunit of laminin 2. In some instances, the targeting moiety disclosed herein binds to the yl -subunit of laminin 2. In some instances, the targeting moiety disclosed herein binds amino acids 2901-3106 of lamin-2. In other instances, the targeting moiety disclosed herein binds to P0 protein (myelin protein zero). In some instances, the targeting moiety disclosed herein binds to an N-terminal extracellular immunoglobulin (Ig)- like domain of P0 protein. In some instances, the targeting moiety disclosed herein binds to the N-linked oligosaccharide in the extracellular domain of the leprosy receptor or any subunits thereof, and / or the addition of sulfate, acyl, and phosphate groups.

[0111] In some instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof which binds to a leprosy receptor. In specific instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof which binds to laminin 2. In specific instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof which binds to the C-terminal region of laminin 2. In some instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof which binds to the a2-subunit of laminin 2. In some instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof which binds to G domain of the a2-subunit of laminin 2. In some instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof which binds to G1 domain of the a2-subunit of laminin 2. In some instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof which binds to G2 domain of the a2-subunit of laminin 2. In some instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof which binds to G3 domain of the a2-subunit of laminin 2. In some instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof which binds to G4 domain of the a2-subunit of laminin 2. In some instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof which binds to G5 domain of the a2-subunit of laminin 2. In some instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof which binds to the 01 -subunit of laminin 2. In some instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof which binds to the yl -subunit of laminin 2. In some instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof which binds amino acids 2901-3106 of lamin-2. In other instances, the targeting moiety disclosed herein is an antibody or antigenbinding fragment thereof which binds to PO protein (myelin protein zero). In some instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof which binds to an N-terminal extracellular immunoglobulin (Ig)-like domain of PO protein. In some instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof which binds to the N-linked oligosaccharide in the extracellular domain of the leprosy receptor or any subunits thereof, and / or the addition of sulfate, acyl, and phosphate groups.In some instances, the targeting moiety disclosed herein is a commercially available antibody that binds to laminin-2. In some instances, the targeting moiety disclosed herein is anti-laminin antibody ABIN7439129. In some instances, the targeting moiety disclosed herein is anti- laminin alpha-2 monoclonal antibody (Thermo Fisher, #CL3450).

[0112] In some instances, the targeting moiety disclosed herein binds to a cell adhesion molecule (Cadm). In some instances, the targeting moiety disclosed herein binds to Cadml. In some instances, the targeting moiety disclosed herein binds to IgV-set domain of Cadml or a portion thereof. In some instances, the targeting moiety disclosed herein binds to IgCl-set domain of Cadml or a portion thereof. In some instances, the targeting moiety disclosed herein binds to Igl-set domain of Cadml or a portion thereof. In specific instances, the targeting moiety disclosed herein binds to Cadm2. In some instances, the targeting moiety disclosed herein binds to IgV-set domain of Cadm2 or a portion thereof. In some instances, the targeting moiety disclosed herein binds to IgCl-set domain of Cadm2 or a portion thereof. In some instances, the targeting moiety disclosed herein binds to Igl-set domain of Cadm2 or a portion thereof. In some instances, the targeting moiety disclosed herein binds to Cadm3. In some instances, the targeting moiety disclosed herein binds to IgV-set domain of Cadm3 or a portion thereof. In some instances, the targeting moiety disclosed herein binds to IgCl-set domain of Cadm3 or a portion thereof. In some instances, the targeting moiety disclosed herein binds to Igl-set domain of Cadm3 or a portion thereof. In some instances, the targeting moiety disclosed herein binds to Cadm4. In some instances, the targeting moiety disclosed herein binds to IgV-set domain of Cadm4 or a portion thereof. In some instances, the targeting moiety disclosed herein binds to IgCl-set domain of Cadm4 or a portion thereof. In some instances, the targeting moiety disclosed herein binds to Igl-set domain of Cadm4 or a portion thereof.

[0113] In some instances, the targeting moiety disclosed herein an antibody or antigen binding fragment thereof which binds to a cell adhesion molecule (Cadm). In some instances, the targeting moiety disclosed herein an antibody or antigen binding fragment thereof which binds to Cadml. In some instances, the targeting moiety disclosed herein an antibody or antigenbinding fragment thereof which binds to IgV-set domain of Cadml or a portion thereof. In some instances, the targeting moiety disclosed herein an antibody or antigen binding fragment thereof which binds to IgCl -set domain of Cadml or a portion thereof. In some instances, the targeting moiety disclosed herein an antibody or antigen binding fragment thereof which binds to Igl-set domain of Cadml or a portion thereof. In specific instances, the targeting moiety disclosed herein an antibody or antigen binding fragment thereof which binds to Cadm2. In some instances, the targeting moiety disclosed herein an antibody or antigen binding fragment thereof which binds to IgV-set domain of Cadm2 or a portion thereof. In some instances, the targeting moiety disclosed herein an antibody or antigen binding fragment thereof which binds to IgCl- set domain of Cadm2 or a portion thereof. In some instances, the targeting moiety disclosed herein an antibody or antigen binding fragment thereof which binds to Igl-set domain of Cadm2 or a portion thereof. In some instances, the targeting moiety disclosed herein an antibody or antigen binding fragment thereof which binds to Cadm3. In some instances, the targeting moiety disclosed herein an antibody or antigen binding fragment thereof which binds to IgV- set domain of Cadm3 or a portion thereof. In some instances, the targeting moiety disclosed herein an antibody or antigen binding fragment thereof which binds to IgCl-set domain of Cadm3 or a portion thereof. In some instances, the targeting moiety disclosed herein an antibody or antigen binding fragment thereof which binds to Igl-set domain of Cadm3 or a portion thereof. In some instances, the targeting moiety disclosed herein an antibody or antigen binding fragment thereof which an antibody or antigen binding fragment thereof which binds to Cadm4. In some instances, the targeting moiety disclosed herein an antibody or antigen binding fragment thereof which binds to IgV-set domain of Cadm4 or a portion thereof. In some instances, the targeting moiety disclosed herein an antibody or antigen binding fragment thereof which binds to IgCl -set domain of Cadm4 or a portion thereof. In some instances, the targeting moiety disclosed herein an antibody or antigen binding fragment thereof which binds to Igl-set domain of Cadm4 or a portion thereof.

[0114] In some instances, the targeting moiety disclosed herein is a commercially available antibody that binds to Cadml. In some instances, the targeting moiety disclosed herein is a commercially available antibody that binds to Cadm2. In some instances, the targeting moiety disclosed herein is a commercially available antibody that binds to Cadm3. In some instances, the targeting moiety disclosed herein is a commercially available antibody that binds to Cadm4. In some instances, the targeting moiety disclosed herein is anti-Cadm4 mAb244 / 5 (NeuroMAB). In some instances, the targeting moiety disclosed herein is anti-humanGliomedin Antibody. In some instances, the targeting moiety disclosed herein is Necl4-Fc antibody from Eshed et al., Neuron, volume 47, issue 2, p215-229. In some instances, the targeting moiety disclosed herein is anti-SynCAM4 Antibody (Biolegand, #833302). In some instances, the targeting moiety disclosed herein is IGSF4C / SynCAM4 Antibody (rndsystems, #MAB41642).

[0115] In some instances, the targeting moiety directs the conjugate to Schwann cells, and the conjugates are internalized upon the binding of the targeting moiety to the cell adhesion moiety or receptor, which is about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, or up to 90 days after contacting the Schwann cell. In some instances, the targeting moiety directs the conjugate to Schwann cells, and the conjugates are internalized upon the binding of the targeting moiety to the receptor, which is within about 24 hours, about 36 hours, about 48 hours, about 60 hours, or up to 90 days after contacting the Schwann cell. In some instances, the targeting moiety disclosed herein directs and internalizes the conjugate disclosed herein more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours after contacting the Schwann cell. In some instances, the targeting moiety disclosed herein directs and internalizes the conjugate disclosed herein less than 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 hours after contacting the Schwann cell.

[0116] In some instances, the drug molecules disclosed herein are released after being internalized into the Schwann cells. In some instances, the drug molecules disclosed herein are released after the conjugates disclosed herein enter a lysosome of the Schwann cells. In some instances, the drug molecules disclosed herein are released to the cytoplasm of the Schwann cells. In some instances, the drug molecules disclosed herein are released to the nucleus of the Schwann cells.

[0117] In some aspects, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof. In some instances, the antibody or antigen binding fragment thereof comprises IgG (Immunoglobulin G), including IgGl, IgG2a IgG2b, IgG3 and IgG4, IgA (Immunoglobulin A), including IgAl and IgA2, IgM (Immunoglobulin M), IgE (Immunoglobulin E), IgD (Immunoglobulin D), fragment antigen-binding (Fab fragment), fragment crystallizable region (Fc region), monovalent Fab’, divalent Fab2, single-chain variable fragment (scFv), diabody, minibody, nanobody, single-domain antibody (sdAb), or camelid antibody or binding fragment thereof. In other instances, the targeting moiety disclosed herein is (scFv)2, Fab, Fab', F(ab')2, Fv, dAb, Fd fragments, diabodies, F(ab')3, disulfide linkedFv, sdAb (VHH or nanobody), CDR (Complementarity-determining region), di-scFv, bi-scFv, tascFv (tandem scFv), triabody, tetrabody, V-NAR domain, Fcab, IgGACH2, DVD-Ig, probody, a DARPin, a Centyrin, an affibody, an affilin, an affitin, an anticalin, an avimer, a Fynomer, a Kunitz domain peptide, a monoclonal antibody, a monobody (or adnectin), a tribody, and a nanofitin and mini-proteins or camelid antibody, or a monospecific, bispecific, trispecific, or a multi-specific antibody or binding fragment or antibody-mimetic thereof. In other instances, the targeting moiety disclosed herein is variants and derivatives of antibodies including antibody functional fragments that retain the ability to bind to the receptor expressed on a Schwann cell. Exemplary functional fragments include Fab fragments (e.g., an antibody fragment that contains the antigen-binding domain and comprises a light chain and part of a heavy chain bridged by a disulfide bond); Fab’ (e.g., an antibody fragment containing a single antigen-binding domain comprising an Fab’ and an additional portion of the heavy chain through the hinge region); F(ab’)2 (e.g., two Fab’ molecules joined by interchain disulfide bonds in the hinge regions of the heavy chains; the Fab’ molecules may be directed toward the same or different epitopes); a bispecific Fab (e.g., a Fab molecule having two antigen binding domains, each of which may be directed to a different epitope); a single chain comprising a variable region, also known as, scFv (e.g., the variable, antigen-binding determinative region of a single light and heavy chain of an antibody linked together by a chain of 10-25 amino acids); a disulfide-linked Fv, or dsFv (e.g., the variable, antigen-binding determinative region of a single light and heavy chain of an antibody linked together by a disulfide bond); a camelized VH (e.g., the variable, antigen-binding determinative region of a single heavy chain of an antibody in which some amino acids at the VH interface are those found in the heavy chain of naturally occurring camel antibodies); a bispecific scFv (e.g., an scFv or a dsFv molecule having two antigen-binding domains, each of which may be directed to a different epitope); a diabody (e.g., a dimerized scFv formed when the VH domain of a first scFv assembles with the VL domain of a second scFv and the VL domain of the first scFv assembles with the VH domain of the second scFv; the two antigen-binding regions of the diabody may be directed towards the same or different epitopes); a triabody (e.g., a trimerized scFv, formed in a manner similar to a diabody, but in which three antigen-binding domains are created in a single complex; the three antigen binding domains may be directed towards the same or different epitopes) ; and a tetrabody (e.g., a tetramerized scFv, formed in a manner similar to a diabody, but in which four antigen-binding domains are created in a single complex; the four antigen binding domains may be directed towards the same or different epitopes). In some aspects, the targeting moietydisclosed herein is an antibody or antigen binding fragment thereof with mono-specificity, bispecificity, tri-specificity, or multi-specificity. In other aspects, the targeting moiety disclosed herein is a combination of one or more of the above-mentioned antibodies or antigen binding fragments thereof.

[0118] In some aspects, the targeting moiety disclosed herein is a ligand molecule. In some instances, the ligand molecule is recognized by Schwann cells and the conjugates disclosed herein are internalized into the Schwann cells. In some instances, the ligand molecule comprises a protein, a peptide, a glycoprotein, a glycan, a carbohydrate moiety, a fatty acid moiety or molecule, a dendrimer, or a synthetic small molecule. In some instances, the ligand molecule described herein binds to laminin 2. In some instances, the ligand molecule described herein binds to P0 protein. In some instances, the ligand molecule described herein binds to gliomedin. In some instances, the ligand molecule described herein binds to Cadml . In some instances, the ligand molecule described herein binds to Cadm2. In some instances, the ligand molecule described herein binds to Cadm3. In some instances, the ligand molecule described herein binds to Cadm4.

[0119] In other aspects, the targeting moiety disclosed herein is a combination of one or more of the above-mentioned antibodies, antigen binding fragments thereof, or ligand molecules.

[0120] In some instances, the targeting moiety disclosed herein comprises at least a portion of neurofascin-186 (NF186) or a derivative thereof. In some instances, the targeting moiety disclosed herein comprises one or more gliomedin-binding domain of NF 186.

[0121] In some instances, the targeting moiety disclosed herein comprises at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, or 60% of full length NF 186 without significantly affecting the binding affinity to gliomedin. In some instances, the targeting moiety disclosed herein comprises no greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of full length NF 186 without significantly affecting the binding affinity to gliomedin. In some instances, the targeting moiety disclosed herein comprises a portion of NF 186 with one or more conservative amino acid substitutions without significantly affecting the binding affinity to gliomedin. In some instances, the targeting moiety disclosed herein comprises a portion of NF 186 with one or more non-conservative amino acid substitutions without significantly affecting the binding affinity to gliomedin. In some instances, the targeting moiety disclosed herein comprises a portion of NF186 with one or more substitution with a basic, an acidic, an aromatic, an aliphatic, an acid amide, a cyclic, or a sulfur- containing amino acid without significantly affecting the binding affinity to gliomedin. In some instances, the targeting moietydisclosed herein comprises a portion of NF 186 with one or more substitution with a standard amino acid or a non-standard amino acid without significantly affecting the binding affinity to gliomedin. In some instances, the targeting moiety disclosed herein comprises a portion of NF 186 with one or more substitution with an alpha, a beta, a gamma, or a delta amino acid without significantly affecting the binding affinity to gliomedin. In some instances, the targeting moiety disclosed herein comprises a portion of NF 186 with one or more substitution with a positively-charged, negatively-charged, or neutral amino acid without significantly affecting the binding affinity to gliomedin. In some instances, the targeting moiety disclosed herein comprises a portion of NF 186 with one or more substitution with a polar or non-polar amino acid without significantly affecting the binding affinity to gliomedin. In some instances, the targeting moiety disclosed herein comprises a portion of NF186 with one or more amino acid deletions without significantly affecting the binding affinity to gliomedin. In some instances, the targeting moiety disclosed herein comprises a portion of NF186 with one or more amino acid insertions without significantly affecting the binding affinity to gliomedin. In some instances, the targeting moiety disclosed herein comprises a portion of NF 186 and an amino acid sequence which is at least partly the reverse of the amino acid sequence of the corresponding portion of NF 186, without significantly affecting the binding affinity to gliomedin. In some instances, the targeting moiety disclosed herein comprises a portion of NF 186, and wherein at least one of the amino acids from the original portion of NF 186 is replaced by a stereoisomer (e.g., D-stereoisomer) of that amino acid, without significantly affecting the binding affinity to gliomedin. In some instances, the targeting moiety disclosed herein comprises a portion of NF186, and is extended at one or both ends thereof with one or more groups, such as D-amino acids, without significantly affecting the binding affinity to gliomedin. In some instances, the targeting moiety disclosed herein comprises NF 186 with a combination of one or more of the above-mentioned modifications without significantly affecting the binding affinity to gliomedin.

[0122] In some instances, the targeting moiety disclosed herein comprises a recombinant NF186 peptide or a derivative thereof. In some instances, the targeting moiety disclosed herein comprises a gliomedin-binding domain of recombinant NF186. In some instances, the targeting moiety disclosed herein comprises at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, or 60% of full length recombinant NF186 without significantly affecting the binding affinity to gliomedin. In some instances, the targeting moiety disclosed herein comprises no greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of full length recombinant NF 186 withoutsignificantly affecting the binding affinity to gliomedin. In some instances, the targeting moiety disclosed herein comprises a portion of recombinant NF 186 with one or more conservative amino acid substitutions without significantly affecting the binding affinity to gliomedin. In some instances, the targeting moiety disclosed herein comprises a portion of recombinant NF 186 with one or more non-conservative amino acid substitutions without significantly affecting the binding affinity to gliomedin. In some instances, the targeting moiety disclosed herein comprises a portion of recombinant NF 186 with one or more substitution with a basic, an acidic, an aromatic, an aliphatic, an acid amide, a cyclic, or a sulfur-containing amino acid without significantly affecting the binding affinity to gliomedin. In some instances, the targeting moiety disclosed herein comprises a portion of recombinant NF 186 with one or more substitution with a standard amino acid or a non-standard amino acid without significantly affecting the binding affinity to gliomedin. In some instances, the targeting moiety disclosed herein comprises a portion of recombinant NF186 with one or more substitution with an alpha, a beta, a gamma, or a delta amino acid without significantly affecting the binding affinity to gliomedin. In some instances, the targeting moiety disclosed herein comprises a portion of recombinant NF 186 with one or more substitution with a positively-charged, negatively - charged, or neutral amino acid without significantly affecting the binding affinity to gliomedin. In some instances, the targeting moiety disclosed herein comprises a portion of recombinant NF186 with one or more substitution with a polar or non-polar amino acid without significantly affecting the binding affinity to gliomedin. In some instances, the targeting moiety disclosed herein comprises a portion of recombinant NF 186 with one or more amino acid deletions without significantly affecting the binding affinity to gliomedin. In some instances, the targeting moiety disclosed herein comprises a portion of recombinant NF186 with one or more amino acid insertions without significantly affecting the binding affinity to gliomedin. In some instances, the targeting moiety disclosed herein comprises a portion of recombinant NF 186 and an amino acid sequence which is at least partly the reverse of the amino acid sequence of the corresponding portion of recombinant NF 186 without significantly affecting the binding affinity to gliomedin. In some instances, the targeting moiety disclosed herein comprises a portion of recombinant NF 186, and wherein at least one of the amino acids from the original portion of recombinant NF 186 is replaced by a stereoisomer (e.g., D-stereoisomer) of that amino acid without significantly affecting the binding affinity to gliomedin. In some instances, the targeting moiety disclosed herein comprises a portion of recombinant NF 186, and is extended at one or both ends thereof with one or more groups, such as D-amino acids, withoutsignificantly affecting the binding affinity to gliomedin. In some instances, the targeting moiety disclosed herein comprises recombinant NF 186 with a combination of one or more of the above-mentioned modifications without significantly affecting the binding affinity to gliomedin.

[0123] In some instances, the targeting moiety disclosed herein binds to a neural-specific extracellular matrix protein that binds to a receptor expressed on Schwann cells. In some instances, the targeting moiety disclosed herein binds to laminin 2. In some instances, the targeting moiety disclosed herein binds to the a2-subunit of laminin 2. In some instances, the targeting moiety disclosed herein binds to G domain of the a2-subunit of laminin 2. In some instances, the targeting moiety disclosed herein binds to G1 domain of the a2-subunit of laminin 2. In some instances, the targeting moiety disclosed herein binds to G2 domain of the a2-subunit of laminin 2. In some instances, the targeting moiety disclosed herein binds to G3 domain of the a2-subunit of laminin 2. In some instances, the targeting moiety disclosed herein binds to G4 domain of the a2-subunit of laminin 2. In some instances, the targeting moiety disclosed herein binds to G5 domain of the a2-subunit of laminin 2. In some instances, the targeting moiety disclosed herein binds to the 01 -subunit of laminin 2. In some instances, the targeting moiety disclosed herein binds to the yl -subunit of laminin 2.

[0124] In some instances, the targeting moiety disclosed herein comprises at least a portion of glycolipid PGL-1 or a derivative thereof. In some instances, the targeting moiety disclosed herein comprises the unique trisaccharide of PGL-1 (3,6-di-O-methylglucose linked a-1— >4 to 2,3-di-O-methylrhamnose linked 0-1 — >2 to 3-O-methylrhamnose).

[0125] In some instances, the targeting moiety disclosed herein comprises at least a portion of ML-LBP21 (also known histone-like protein / Hlp) or a derivative thereof. In some instances, the targeting moiety disclosed herein comprises a laminin-2-binding domain of ML-LBP21. In some instances, the targeting moiety disclosed herein comprises at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, or 60% of full length ML-LBP21 without significantly affecting the binding affinity to laminin-2. In some instances, the targeting moiety disclosed herein comprises no greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of full length ML-LBP21 without significantly affecting the binding affinity to laminin-2. In some instances, the targeting moiety disclosed herein comprises a portion of ML-LBP21 with one or more conservative amino acid substitutions without significantly affecting the binding affinity to laminin-2. In some instances, the targeting moiety disclosed herein comprises a portion of ML- LBP21 with one or more non-conservative amino acid substitutions without significantlyaffecting the binding affinity to laminin-2. In some instances, the targeting moiety disclosed herein comprises a portion of ML-LBP21 with one or more substitution with a basic, an acidic, an aromatic, an aliphatic, an acid amide, a cyclic, or a sulfur-containing amino acid without significantly affecting the binding affinity to laminin-2. In some instances, the targeting moiety disclosed herein comprises a portion of ML-LBP21 with one or more substitution with a standard amino acid or a non-standard amino acid without significantly affecting the binding affinity to laminin-2. In some instances, the targeting moiety disclosed herein comprises a portion of ML-LBP21 with one or more substitution with an alpha, a beta, a gamma, or a delta amino acid without significantly affecting the binding affinity to laminin-2. In some instances, the targeting moiety disclosed herein comprises a portion of ML-LBP21 with one or more substitutions with a positively-charged, negatively-charged, or neutral amino acid without significantly affecting the binding affinity to laminin-2. In some instances, the targeting moiety disclosed herein comprises a portion of ML-LBP21 with one or more substitution with a polar or non-polar amino acid without significantly affecting the binding affinity to laminin-2. In some instances, the targeting moiety disclosed herein comprises a portion of ML-LBP21 with one or more amino acid deletions without significantly affecting the binding affinity to laminin- 2. In some instances, the targeting moiety disclosed herein comprises a portion of ML-LBP21 with one or more amino acid insertions without significantly affecting the binding affinity to laminin-2. In some instances, the targeting moiety disclosed herein comprises a portion of ML- LBP21 and an amino acid sequence which is at least partly the reverse of the amino acid sequence of the corresponding portion of ML-LBP21, without significantly affecting the binding affinity to laminin-2. In some instances, the targeting moiety disclosed herein comprises a portion of ML-LBP21, and wherein at least one of the amino acids from the original portion of ML-LBP21 is replaced by a stereoisomer (e.g., D-stereoisomer) of that amino acid, without significantly affecting the binding affinity to laminin-2. In some instances, the targeting moiety disclosed herein comprises a portion of ML-LBP21, and is extended at one or both ends thereof with one or more groups, such as D-amino acids, without significantly affecting the binding affinity to laminin-2. In some instances, the targeting moiety disclosed herein comprises ML-LBP21 with a combination of one or more of the above-mentioned modifications without significantly affecting the binding affinity to laminin-2.

[0126] In some instances, the targeting moiety disclosed herein comprises a recombinant ML- LBP21 peptide or a derivative thereof. In some instances, the targeting moiety disclosed herein comprises a laminin-2-binding domain of recombinant ML-LBP21. In some instances, thetargeting moiety disclosed herein comprises at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, or 60% of full length recombinant ML-LBP21 without significantly affecting the binding affinity to laminin-2. In some instances, the targeting moiety disclosed herein comprises no greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of full length recombinant ML-LBP21 without significantly affecting the binding affinity to laminin-2. In some instances, the targeting moiety disclosed herein comprises a portion of recombinant ML- LBP21 with one or more conservative amino acid substitutions without significantly affecting the binding affinity to laminin-2. In some instances, the targeting moiety disclosed herein comprises a portion of recombinant ML-LBP21 with one or more non-conservative amino acid substitutions without significantly affecting the binding affinity to laminin-2. In some instances, the targeting moiety disclosed herein comprises a portion of recombinant ML-LBP21 with one or more substitution with a basic, an acidic, an aromatic, an aliphatic, an acid amide, a cyclic, or a sulfur-containing amino acid without significantly affecting the binding affinity to laminin-2. In some instances, the targeting moiety disclosed herein comprises a portion of recombinant ML-LBP21 with one or more substitution with a standard amino acid or a nonstandard amino acid without significantly affecting the binding affinity to laminin-2. In some instances, the targeting moiety disclosed herein comprises a portion of recombinant ML-LBP21 with one or more substitution with an alpha, a beta, a gamma, or a delta amino acid without significantly affecting the binding affinity to laminin-2. In some instances, the targeting moiety disclosed herein comprises a portion of recombinant ML-LBP21 with one or more substitution with a positively-charged, negatively-charged, or neutral amino acid without significantly affecting the binding affinity to laminin-2. In some instances, the targeting moiety disclosed herein comprises a portion of recombinant ML-LBP21 with one or more substitution with a polar or non-polar amino acid without significantly affecting the binding affinity to laminin-2. In some instances, the targeting moiety disclosed herein comprises a portion of recombinant ML-LBP21 with one or more amino acid deletions without significantly affecting the binding affinity to laminin-2. In some instances, the targeting moiety disclosed herein comprises a portion of recombinant ML-LBP21 with one or more amino acid insertions without significantly affecting the binding affinity to laminin-2. In some instances, the targeting moiety disclosed herein comprises a portion of recombinant ML-LBP21 and an amino acid sequence which is at least partly the reverse of the amino acid sequence of the corresponding portion of recombinant ML-LBP21 without significantly affecting the binding affinity to laminin-2. In some instances, the targeting moiety disclosed herein comprises a portion of recombinant ML-LBP21, and wherein at least one of the amino acids from the original portion of recombinant ML-LBP21 is replaced by a stereoisomer (e.g., D-stereoisomer) of that amino acid without significantly affecting the binding affinity to laminin-2. In some instances, the targeting moiety disclosed herein comprises a portion of recombinant ML-LBP21, and is extended at one or both ends thereof with one or more groups, such as D-amino acids, without significantly affecting the binding affinity to laminin-2. In some instances, the targeting moiety disclosed herein comprises recombinant ML-LBP21 with a combination of one or more of the above-mentioned modifications without significantly affecting the binding affinity to laminin-2.

[0127] In some aspects, the targeting moiety disclosed herein comprises a combination of one or more of the above-mentioned molecules.Drug Molecules

[0128] In some aspects, the drug molecules disclosed herein modify the expression of a disease- associated molecule, such as modifying expression level of a gene. In specific aspects, the overexpression of the disease-associated molecule leads to the disease. Accordingly, in some cases, the drug molecules disclosed herein reduce the transcription / RNA level of the disease- associated molecule by at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. Accordingly, in some cases, the drug molecules disclosed herein reduce the transcription / RNA level of the disease-associated molecule to a level of those of healthy individuals. Accordingly, in other cases, the drug molecules disclosed herein reduce the translation / protein level of the disease-associated molecule by at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. Accordingly, in some cases, the drug molecules disclosed herein reduce the translation / protein level of the disease-associated molecule to a level of those of healthy individuals.

[0129] In specific aspects, the lack of the disease-associated molecule leads to the disease. Accordingly, in some cases, the drug molecules disclosed herein increase the expression level of the disease-associated molecule, such as increase expression level of a gene, for example increase the transcription / RNA level of the disease-associated molecule by at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. Accordingly, in some cases, the drug molecules disclosed herein increase the transcription / RNA level of the disease-associated molecule to a level of those of healthy individuals. Accordingly, in other cases, the drug molecules disclosed herein increase the translation / protein level of the disease-associated molecule by at least 1%, 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. Accordingly, in some cases, the drug molecules disclosed herein increase the translation / protein level of the disease-associated molecule to a level of those of healthy individuals.

[0130] In some embodiments, the drug molecules disclosed herein modify the expression of a disease-associated molecule, such as modifying expression level of a gene. In certain aspects, the drug molecule comprises RNA molecule such as tRNA, rRNA and mRNA, coding (cRNA), noncoding RNA (ncRNA), micro RNA (miRNA), small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), small-interfering RNA (siRNA), PlWI-interacting RNA (piRNA), short hairpin RNA (shRNA), microRNA (miRs), a double-stranded RNAi molecule, a singlestranded antisense oligonucleotide, antisense oligonucleotides (ASOs) and antiparallel triplex forming oligonucleotides. In specific embodiments, post-transcriptional gene modulation is exerted via RNA interference (RNAi) as a drug molecule. The primary problem with using RNAi in gene modulation is drug delivery to target cells. The drug conjugate of the present invention enables targeted delivery of the RNAi to the desired cell or tissue or organ to manipulate specific gene expression for achieving a therapeutic effect.

[0131] In some embodiments, the RNA molecules used herein as a drug molecule within the disclosed antibody-drug conjugate (e.g. siRNA) are stabilized or modified to improve stabilization and preserve the integrity of RNA. Stabilization and / or modifications of RNA molecules (e.g. siRNA) within the scope of the present invention include, but are not limited to:

[0132] Stabilization of small interfering RNA (siRNA)- Stabilization of siRNA is crucial to improve its stability, bioavailability, and efficacy in therapeutic applications. siRNA molecules are prone to degradation by nucleases, rapid renal clearance, and immune system activation. Several chemical and structural modifications can be employed to stabilize siRNA (see jnanobiotechnology.biomedcentral.com ), including

[0133] 1. Chemical Modifications to Sugar Moiety

[0134] 2'-O-Methyl (2'-0Me): A common modification that improves resistance to nucleases and reduces immunogenicity.

[0135] 2' -Fluoro (2'-F): Enhances nuclease resistance and maintains high binding affinity to the RNA-induced silencing complex (RISC).

[0136] 2'-O-Methoxyethyl (2'-M0E): Further improves stability and binding affinity.

[0137] Locked Nucleic Acids (LNAs): Constrain the ribose ring, improving thermal stability and nuclease resistance.

[0138] 2. Backbone Modifications

[0139] Phosphorothioate Linkages: Substitute one of the non-bridging oxygen atoms in the phosphate backbone with sulfur, increasing nuclease resistance and improving serum stability.

[0140] Boranophosphate: Replace one of the phosphate oxygens with borane, enhancing stability and activity.

[0141] 3. Base Modifications

[0142] Modified bases can enhance the affinity of siRNA for the target mRNA and reduce off- target effects. Examples include methylated bases or incorporation of non-natural bases.

[0143] 4. End Modifications

[0144] Capping at 3' and 5' Ends: Adding modifications like inverted deoxythymidine (idT) or other chemical groups to protect against exonuclease activity.

[0145] Addition of Conjugates: Adding lipid moieties (e.g., cholesterol), peptides, or polymers (e.g., polyethylene glycol) to improve cellular uptake and reduce degradation.

[0146] 5. Conjugation with Stabilizing Agents

[0147] PEGylation: Attaching polyethylene glycol to siRNA enhances circulation time and reduces renal clearance.

[0148] Lipid or Polymer Conjugates: Facilitate entry into cells and provide steric hindrance against enzymatic degradation.

[0149] 6. Use of Double-Stranded RNA-Binding Proteins

[0150] Incorporation of certain stabilizing proteins or designing siRNA to interact with RNA- binding domains for protection against degradation.

[0151] 7. Structural Modifications

[0152] Asymmetric siRNA (asiRNA): Design one strand shorter than the other to reduce off- target effects and increase stability.

[0153] Blunt-End Modifications: Add non-natural linkages at the blunt ends to improve stability.

[0154] In other aspects, the drug molecules disclosed herein modify the activity of a disease- associated molecule. In specific aspects, the hyperactivity of the disease-associated molecule leads to the disease. Accordingly, in some cases, the drug molecules disclosed herein reduce the activity of the disease-associated molecule by at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. Accordingly, in some cases, the drug molecules disclosed herein reduce the activity of the disease-associated molecule to a level of those of healthy individuals. In specific aspects, the hypoactivity of thedisease-associated molecule leads to the disease. Accordingly, in some cases, the drug molecules disclosed herein increase the activity of the disease-associated molecule by at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. Accordingly, in some cases, the drug molecules disclosed herein increase the activity of the disease-associated molecule to a level of those of healthy individuals.

[0155] In some instances, the drug molecule comprises a drug or a precursor form of the drug (a drug precursor or a prodrug). In some instances, the drug molecule comprises a peptide. In some instances, the drug molecule comprises a protein. Any suitable peptide or protein may be used as a drug molecule to modify the expression or activity of the disease-associated molecule, as described herein. In some cases, the drug molecule comprises an enzyme. In some cases, peptides or proteins may be produced, synthesized, and / or derivatized using several methodologies, e.g. phage displayed peptide libraries, one-bead one-compound peptide libraries, directed evolution, or positional scanning synthetic peptide combinatorial libraries. In some instances, the drug molecule comprises peptide or protein of about 2-25 amino acids, about 2-20 amino acids, about 2-15 amino acids, about 2-10 amino acids, or about 2-5 amino acids. The peptide or protein may comprise naturally-occurring amino acids, e.g. cysteine, alanine, or non-naturally-occurring or modified amino acids. Non-naturally occurring amino acids include 3 -amino acids, homo-amino acids, proline derivatives, 3 -substituted alanine derivatives, linear core amino acids, N-methyl amino acids, and others known in the art. In some cases, the peptide may be linear; in other instances, the peptide may be cyclic, e.g. bicyclic.

[0156] According to some embodiments of the present invention, the conjugated drug molecules disclosed herein are small molecular weight (SMW), also referred to as small molecule (SM), compounds that inhibit or modulate the activity of specific enzymes, proteins, kinases and / or phosphatases or any cell activity-related moiety or any protein-specific modulating agent. In some embodiments, such enzymes, proteins, kinases and / or phosphatases are implicated in many cellular processes, such as proliferation, differentiation and oncogenic transformation. These proteins are crucial regulators of intracellular signaling pathways mediating multiple cellular activities. Therefore, alterations in kinases and phosphatases functionality may be involved in potency of cancer cells (e.g. metastasis or metastatic disease). Targeting enzymes, proteins, kinases and / or phosphatases by conjugated SMW drug molecules of the present invention enable effective anti-cancer therapy. In some instances, such SMWs may be RKIs, potent inhibitors of the Rho-associated ROCK kinases, highly involved in cancercell migration and invasion. According to some embodiments, SMW inhibitors of kinases include, in a non limiting manner, calcium / calmodulin, phospholipids, cAMP and cGMP, growth factors, cytokines, and toxins. In some embodiments, SMW inhibitors of phosphatases, include, in a non limiting manner, okadaic acid, calyculin, tautomycin, certain vanadium compounds, and other phosphatase inhibitors. In some instances, a non limiting list of kinases and phosphatases SMW inhibitors may include, AKT (Protein Kinase B) Inhibitors, MAP Kinase Inhibitors, Protein Kinase C Inhibitors, Protein Phosphatase Inhibitors, Protein Tyrosine Kinase Inhibitors, Rho Kinase (ROCK) Inhibitors and / or any other Kinase / Phosphatase Inhibitors known in the art.

[0157] In some instances, the drug molecule comprises a small molecule. In some instances, the small molecule is a modulator of the disease-associated molecule. In some instances, the small molecule is an agonist of the disease-associated molecule. In some instances, the small molecule is an antagonist of the disease-associated molecule. In some instances, the small molecule is an inhibitor of the disease-associated molecule. In some instances, the small molecule is a protein degrader molecule, for targeted protein degradation of a Protein of Interest (e.g. a disease associated molecule), for example, Mancarella, C., et al., Int J Mol Sci., 2023, 24, 16346, is incorporated herein by reference in its entirety.

[0158] In some instances, the small molecule is a cytotoxic agent. The term “cytotoxic agent” or "cytotoxic effect" refers to a substance that inhibits or prevents the expression activity of cells, function of cells and / or causes destruction of cells. It further refers to a substance or process that can damage cells or cause them to die. The term is intended to include radioactive isotopes, chemotherapeutic agents, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof. Examples of cytotoxic agents include, but are not limited to auristatins (e.g., auristatin e, auristatin f, MMAE and MMAF), auromycins, maytansinoids, calicheamicins, pyrrolobenzodiazepines, topoisomerase inhibitors, a-Amanitin, epothilones, ricin, ricin A- chain, combretastatin, duocarmycins, dolastatins, doxorubicin, daunorubicin, taxols, cisplatin, ccl065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracin dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, alpha-sarcin, gelonin, mitogellin, restrictocin, phenomycin, enomycin, curicin, crotin, calicheamicin, Saponaria officinalis inhibitor, and glucocorticoid and other chemotherapeutic agents, as well as radioisotopes such as At211, 1131, 1125, Y90, Rel86, Rel88, Sml53, Bi212 or 213 , P32 and radioactive isotopesof Lu including Lui 77. Antibodies may also be conjugated to an anti-cancer pro-drug activating enzyme capable of converting the pro-drug to its active form. The term "cytotoxicity" refers to the quality of being toxic to cells.

[0159] It is within the scope of the present invention that, the drug molecule as herein disclosed is a cytotoxic agent or a cell death inducing molecule, which is used and targeted for conferring a cytotoxic effect to cancerous or tumor cells for example, in cancer related disorders such as in neurofibromatosis, e.g. schwannoma or schwannomatosis, that causes multiple nerve sheath tumors and other Schwann cell-related oncologic indications.

[0160] According to some embodiments of the present invention, the drug molecule comprises maytansinoids, calicheamicins, pyrrolobenzodiazepines, duocarmycins, topoisomerase inhibitors, a-Amanitin, epothilones, cell death inducing molecules, cytotoxic agent, and / or any combination thereof.

[0161] According to some embodiments, the conjugated drug molecules disclosed herein are toxin molecules (e.g. cytotoxic agents) that cause cytotoxic effect such as targeted cell death. In this case the cell death is induced by the conjugated drug molecule disclosed herein which may encompass a conjugated toxin. It is herein acknowledged that one of the major drawbacks of many of the currently used cancer drugs are off-target effects. Targeted delivery is one method to minimize such undesirable and detrimental events. To actively target cancer cells (e.g. in Schwann cell-related oncologic indications), the inventors of the present invention developed a conjugate of a cell death inducing protein (or a cytotoxic agent) with a targeting moiety specific to a cell adhesion moiety or receptor expressed or overexpressed by rapidly dividing cancer cells (e.g. a specific type of cancer cells). In some instances, the cell death inducing proteins are toxins that are cytopathic to the target cells, e.g. through lytic or non- lytic mechanisms by inducing necrosis or apoptosis. According to some aspects, cell death types include apoptosis, autophagy, necrosis, and entosis. According to further aspects, toxins that can induce cell death include, but are not limited to, autophagic cell death toxins, including cytostatics, ionophores, and oxidants inducing autophagic cell death; ER stress is known to be able to cause apoptosis as well as autophagic cell death; TCDD and cadmium can induce cell death by either apoptosis or autophagy. In some instances, cell death is caused by extrinsic pathways, generally initiated by the activation of death receptors through the interaction between their natural ligands or by inducing death receptor clusterization.

[0162] It is within the scope that cell death receptors include cell surface receptors that belong to the tumor necrosis factor (TNF) super family and interact with their ligands to form deathreceptor complexes, including Fas (CD95 / Apol) / Fas Ligand (CD95 ligand), TNF receptor 1 (p55) / TNF and lymphotoxin, TRAMP (WSL-1 / Apo3 / DR3 / LARD) / TWEAK (Apo3 ligand), TRAIL-R1 (DR4) / TRAIL (Apo2 ligand), and TRAIL-R2 (DR5 / Apo2 / KILLER) / TRAIL. According to some aspects, without wishing to be bound by theory, upon extrinsic activation, the intracellular death domain (DD) of death receptors associates with an adaptor protein called Fas-associated death domain (FADD) directly or indirectly via the TNF receptor-associated death domain. The death receptor associated intracellular FADD interacts with pro-caspase-8, a typical initial caspase, to form a death-inducing signaling complex required for caspase- 8 activation. According to further aspects, during the process of apoptosis, there is, in general, a reduction of mitochondrial transmembrane potential followed by the release of cytochrome c, which binds to Apaf-1 and promotes caspase-9 and caspase-3 activation. It is herein acknowledged that the central role of mitochondria in apoptosis may be via an intrinsic pathway. Pro-apoptotic Bax and Bid, the members of the Bcl-2 family with pro-apoptotic roles, translocate to the mitochondria and disrupt the membrane integrity, resulting in cytochrome c release from mitochondria to cytoplasm. The induction of mitochondrial transmembrane permeabilization (MTP) resulted from Bax or truncated Bid (activated by caspase-8 from the extrinsic pathway) forms pores in the outer membrane directly or by interacting with the permeability of the transition pore complex. In contrast, anti-apoptotic Bcl-2 and Bcl-xL protect these effects by maintaining the MTP through the inhibition of Bax or other pro-apoptotic factors. The loss of balance of Bcl-2 / Bax is believed to contribute to the progression of apoptosis.

[0163] In some instances, the drug molecule comprises an oligonucleotide. In some instances, oligonucleotide comprises a double-stranded RNAi molecule or a single-stranded antisense oligonucleotide. In specific instances, the drug molecule comprises a small interfering RNA (siRNA), a microRNA (miRNA), an inhibitory double stranded RNA (dsRNA), a small or short hairpin RNA (shRNA), a piwi-interacting RNA (piRNA), a heterogeneous nuclear RNA (hnRNA), a small nuclear RNA (snRNA), or an enzymatically-prepared siRNA (esiRNA) or the precursors thereof. In other instances, the drug molecule comprises a nucleic acid disclosed in Sandy et al., Mammalian RNAi: a practical guide, BioTechniques, VOL. 39, NO. 2 REVIEW, which is incorporated by reference herein in its entirety.

[0164] In some instances, the disease-associated molecule is antiganglioside antibodies. Accordingly, the drug molecule disclosed herein modifies antiganglioside antibodies. In some cases, antiganglioside antibodies are associated with the Guillain-Barre syndrome. TheGuillain-Barre syndrome is a post-infectious immune-mediated neuropathy, which results from the autoimmune destruction of nerves in the peripheral nervous system. The syndrome may include muscle movement as well as those that transmit pain, temperature and touch sensations. This can result in muscle weakness and loss of sensation in the legs and / or arms. Antiganglioside antibodies may be involved in the pathogenesis of the Guillain-Barre syndrome.

[0165] In some instances, the disease-associated molecule is a subunit of SWI / SNF protein complexes, which is encoded by gene SMARCB1. In some instances, the disease-associated molecule is LZTR1 protein, which is encoded by LZTR1 gene. Accordingly, the drug molecule disclosed herein modifies the subunit of SWI / SNF protein complexes. Accordingly, the drug molecule disclosed herein modifies LZTR1 protein. In some cases, the subunit of SWI / SNF protein complexes is associated with schwannomatosis. In some cases, LZTR1 protein is associated with schwannomatosis. Schwannomatosis is a type of neurofibromatosis that causes multiple nerve sheath tumors. It is within the scope of the present invention that usage of a small molecule as a drug molecule within the drug conjugate as herein disclosed is provided to target disease associated molecules of Schwannomatosis.

[0166] In some instances, the drug molecule is immunoglobulin, and the associated disease is chronic inflammatory demyelinating polyneuropathy (CIDP). CIDP is a rare disorder of the peripheral nerves characterized by gradually increasing symmetrical motor and sensory loss and weakness associated with loss of deep tendon reflexes. It is caused by damage to Schwann cells. The gradual onset of CIDP can delay diagnosis by several months or even years, resulting in significant nerve damage that may limit and delay the response to therapy. Most individuals may require long term treatment; nearly a third of CIDP patients may progress to wheelchair dependence if left untreated. Early recognition and proper treatment are critical in helping patients avoid a significant amount of disability.

[0167] In some instances, the drug molecules of the present invention are small molecules that can modulate the course of the disease of Neurofibromas. Reference is now made to Neurofibromas, which are the most common peripheral nerve sheath tumor and are often found either by patients or during routine skin exams. These lesions appear as soft, skin-colored papules or small subcutaneous nodules. There are three main types of neurofibromas: localized, diffuse, and plexiform. The majority of neurofibromas are sporadically-occurring and localized and have an extremely low risk of malignant transformation. However, the plexiform type is pathognomonic for neurofibromatosis type 1 and carries an increased risk of malignanttransformation. Literature suggests the contribution of ErbB family receptors in neurofibroama progression. In particular, certain transmembrane receptor tyrosine kinases (RTKs) of the Epidermal growth factor receptor family B (ErbB), such as EGFR, ErbB2, and ErbB3, can serve as potential therapeutic targets in human neurofibromas. Furthermore, Uncontrolled RTK signals lead to cell growth disorders and cancer, which provided the rationale for the development of strategies for the prevention and interception of RTK signaling as a way to treat Neurofibromas. An additional number of RTKs have been shown to be associated with neurofibromas, such platelet-derived growth factor (PDGF), fibroblasts growth factor (FGF), insulin-like growth factor- 1, and vascular endothelial growth factor (VEGF). Additionally, nrPTKs, including Src, BCR-ABL, and the JAK / STAT pathway, are not receptors per se. They generally do not have an extracellular domain, but are coupled to the cell membrane or exist in the cytoplasm, and their tyrosine kinase activity can be activated after binding to the activated receptor. The activation of nrPTKs and downstream signal transduction pathways can promote cell proliferation, cell apoptosis resistance, and oncogenesis. Further, over-activation of the Src pathway has been found in NF2. Therefore, direct and specific SMW compound inhibition of these kinase domain can serve as potential therapeutic pathway to neurofibromas.Coupling Target Moieties and Drug Molecules

[0168] In certain instances, the targeting moieties disclosed herein can be coupled or conjugated to the drug molecules disclosed herein directly (e.g., through a covalent or non- covalent linkage), or through a linker (e.g., a cleavable linker versus a non-cleavable linker, or a peptide linker versus a non-peptide linker), or through an amino acid or other functional group.

[0169] The term "linker" as used herein may generally refers to a compound, or pharmaceutically acceptable solvate, stereoisomer, or derivative thereof, comprising a binding agent linked to at least one pay load moiety (or drug molecule) via e.g. a covalent linker, wherein the said covalent linker is bonded directly or indirectly to each of the binding agent and the payload moiety.

[0170] As used herein, the term "binding agent" refers to a bioconjugation moiety or area. Bioconjugation generally means a specialized area of chemistry that focuses on linking biomolecules to one another or to other types of molecules, such as drugs, fluorescent probes, or nanoparticles. It is herein acknowledged that this process is integral to various fields, including diagnostics, therapeutics, and biomaterials development. Effective bioconjugation relies on reactions that are highly specific, operate under mild conditions to preserve thebiomolecule's function, and result in stable products suitable for biological environments. Common functional groups targeted in bioconjugation include primary amines in lysine residues, thiols in cysteine residues, carboxyl groups, and hydroxyl groups. For example, maleimides are widely used to selectively react with thiols, forming stable thioether bonds. Other strategies include click chemistry, which involves azide-alkyne cycloaddition for bio- orthogonal labeling, and PEGylation, where polyethylene glycol is attached to proteins or drugs to enhance their solubility and stability (see for example Kalia., J et.al. Curr. Org. Chem, 2010 Jan; 14(2): 138-147 doi: 10.2174 / 138527210790069839, incorporated by its entirety).

[0171] According to some aspects of the present invention, the linker is a cleavable linker, a non-cleavable linker, covalently bound linker such as by chemical conjugation, linkers based on non-covalent interactions, a carbohydrate linker, a peptide linker, a glycan linker, or a nonpeptide linker. Examples for such linkers include, but not limited to, Hydrazone Linkers, Disulfide Linkers, Peptide Linkers (Valine-citrulline (Val-Cit) and Alanine-alanine (Ala- Ala)), Beta-Glucuronide Linkers, Thioether Linkers (such as SMCC (succinimidyl-4-(N- maleimidomethyl)cyclohexane-l -carboxylate)), PEG Linkers, Conditional Linkers (such as Azobenzene Linkers and Diselenide Linkers).

[0172] In some instances, the targeting moieties disclosed herein and the drug molecules disclosed herein are connected via a non-covalent interaction. In specific cases, the non- covalent interaction comprises a biotin / avidin interaction. Examples of connecting connect the targeting moiety and the drug molecule include, but are not limited to, the use of biotin and avidin or streptavidin (see, e.g., U.S. Pat. No. 4,885,172 A), typical biotin / avidin alternatives (e.g., FITC / anti-FITC (see, e.g., Harmer and Samuel (1989) J. Immunol. Meth. 122(1): 115- 221), di oxigenin / anti-di oxigenin, and the like.

[0173] In other instances, the targeting moieties disclosed herein and the drug molecules disclosed herein are connected via a covalent interaction. In specific cases, the targeting moieties disclosed herein and the drug molecules disclosed herein are connected via chemical conjugation. Traditional chemical conjugation using, for example, bifunctional coupling agents such as glutaraldehyde, diimide esters, aromatic and aliphatic diisocyanates, bis-p-nitrophenyl esters of dicarboxylic acids, aromatic disulfonyl chlorides and bifunctional arylhalides such as l,5-difluoro-2,4-dinitrobenzene; p,p’-difluoro m,m’ -dinitrodiphenyl sulfone, sulfhydrylreactive maleimides, and the like.

[0174] In some cases, the chemical conjugation of the targeting moieties disclosed herein and the drug molecules disclosed herein is via a non-cleavable linker or via a cleavable linker.

[0175] In some instances, the cleavable linkers are generally cleavable only intracellularly and are preferably stable in extracellular environments, e.g. extracellular to the Schwann cells. In specific cases, the cleavable linker disclosed herein comprises a chemically cleavable linkers. In specific cases, the cleavable linker disclosed herein comprises an enzymatically cleavable linker.

[0176] A number of different chemically cleavable linkers are known to those of skill in the art (see, e.g., U.S. Pat. Nos. 4,618,492; 4,542,225, and 4,625,014). In some specific instances, the chemically cleavable linker is pH-sensitive linker. In some cases, the pH-sensitive linker disclosed herein is readily degrades in high pH environments. In some cases, the pH-sensitive linker disclosed herein is readily degrades in low pH environments. In some instances, the pH- sensitive linker may be cleaved at a pH in a range of 4 to 6. In some instances, the pH-sensitive linker comprises a hydrazone or cyclic acetal. In some instances, the pH-sensitive linker is cleaved within an endosome or a lysosome. In other specific instances, the chemically cleavable linker is glutathione-sensitive linker. In some cases, the glutathione-sensitive linker is cleaved by an disulfide exchange reaction with a glutathione species inside a cell. In some instances, the disulfide moiety further comprises at least one amino acid, e.g. a cysteine residue. Other illustrative chemically cleavable linkers include, but are not limited to, acid-labile linkers, disulfide linkers, and the like. Acid-labile linkers are designed to be stable at pH levels encountered in the blood, but become unstable and degrade when the low pH environment in lysosomes is encountered, an acid-labile linker comprising a moiety selected from the group consisting of a hydrazone, an acetal, a cis-aconitate amide, and a silyl ether. Acid-sensitive linkers include, but are not limited to hydrazones, acetals, cis-aconitate-like amides, and silyl ethers (see, e.g., Perez et al. (2013) Drug Discov. Today, 1-13). Hydrazones are easily synthesized and have a plasma half-life of 183 hours at pH 7 and 4.4 hours at pH 5, indicating that they are selectively cleavable under acidic conditions such as those found in the lysosome (see, e.g., Doronina et al. 92013) Nat. Biotechnol. 21(7): 778-784). Disulfide bridges are cleavable linkers that take advantage of the cellular reducing environment (see, e.g., Saito et al. (2013) Adv. Drug Deliv. Rev. 55(2): 199-215). After internalization and degradation, disulfide bridges can release drugs in the lysosome.

[0177] Enzymatically cleavable linkers are selected to be cleaved by an enzyme (e.g., a protease). In some instances, protease-cleavable linkers are typically designed to be stable in blood / plasma, but are rapidly cleaved in lysosomes by lysosomal enzymes. The most popular enzymatic cleavage sequence is the dipeptide valine-citrulline, combined with a self-immolative linker p-aminobenzyl alcohol (PAB). Cleavage of an amide-linked PAB triggers a 1,6-elimination of carbon dioxide and concomitant release of the free drug in parent amine form (see, e.g., Burke et al. (2009) Bioconjug. Chem. 20(6): 1242-1250). In some specific instances, the cleavable linker is a protease-sensitive linker. The protease-sensitive linker described herein may comprise a sequence cleavable by a lysosomal protease and / or an endosomal protease. In some cases, the protease-sensitive linker typically comprises peptide sequences and may be 2-10 amino acids, about 2-5 amino acids, about 5-10 amino acids, about 10 amino acids, about 5 amino acids, about 3 amino acids, or about 2 amino acids in length. In some instances, the protease-sensitive linker comprises naturally-occurring amino acids, e.g. cysteine, alanine, or non-naturally-occurring or modified amino acids. Non-naturally occurring amino acids include 3-amino acids, homo-amino acids, proline derivatives, 3-substituted alanine derivatives, linear core amino acids, N-methyl amino acids. In some instances, the proteasesensitive linker comprises a valine-citrulline or alanine-citrulline dipeptide sequence. In some instances, the protease-sensitive linker can be cleaved by a lysosomal protease, e.g. cathepsin B, and / or an endosomal protease.

[0178] In some instances, the targeting moiety and the drug molecule are covalently linked to each other via a non-cleavable linker. Generally, the non-cleavable linker cannot be readily degraded in a cellular or physiological environment. In some instances, the non-cleavable linker described herein comprises an optionally substituted alkyl group, wherein the substitutions may include halogens, hydroxyl groups, oxygen species, and other common substitutions. In some instances, the non-cleavable linker described herein comprises an optionally substituted alkyl, an optionally substituted alkylene, an optionally substituted arylene, a heteroarylene, a peptide sequence comprising at least one non-natural amino acid, a truncated glycan, a sugar or sugars that cannot be enzymatically degraded, an azide, an alkyne-azide, a peptide sequence comprising a LPXT sequence, a thioether, a biotin, a biphenyl, repeating units of polyethylene glycol or equivalent compounds, acid esters, acid amides, sulfamides, and / or an alkoxy-amine linker. In some instances, sortase-mediated ligation is utilized to covalently link the drug molecule comprising a LPXT sequence to the targeting moiety comprising a (G). In other instances, sortase-mediated ligation is utilized to covalently link the targeting moiety comprising a LPXT sequence to the drug molecule comprising a (G)sequence (see, e.g. Proft T. Sortase-mediated protein ligation: an emerging biotechnology tool for protein modification and immobilization. Biotechnol Lett. 2010, 32(1): 1-10, which is incorporated by reference herein in its entirety). In some instances, the non-cleavable linkers described herein comprisesa substituted alkylene, an optionally substituted alkenylene, an optionally substituted alkynylene, an optionally substituted cycloalkylene, an optionally substituted cycloalkenylene, an optionally substituted arylene, an optionally substituted heteroarylene further comprising at least one heteroatom selected from N, O, and S; an optionally substituted heterocyclylene further comprising at least one heteroatom selected from N, O, and S; an imino, an optionally substituted nitrogen species, an optionally substituted oxygen species O, an optionally substituted sulfur species, or a poly(alkylene oxide), e.g. polyethylene oxide or polypropylene oxide.

[0179] In some cases, the linkers described herein are peptide linkers. In various instances, the peptide linker is relatively short, typically about 20 amino acids or less, about 15 amino acids or less, about 10 amino acids or less, about 8 amino acids or less, about 5 amino acids or less, about 3 amino acids or less, or is a single amino acid. In some cases, the peptide linker disclosed herein comprises an amino acid sequence cleavable by a protease. In some cases, the peptide linker disclosed herein comprises an amino acid sequence cleavable by a cathepsin. In some cases, the peptide linker disclosed herein comprises a dipeptide valine-citrulline (Val-Cit) (see U.S. Pat. No. 6,214,345, incorporated herein by reference in its entirety) or Phe-Lys. A library of dipeptide linkers was screened by Debowchik and co-workers to measure the rate of doxorubicin release by enzymatic hydrolysis (see, e.g., Dubowchik et al. (2002) Bioconjug. Chem. 13(4): 855-869; Dubowchik et al. (2002) Bioorg. Med. Chem. Lett. 12(11): 1529-1532). They found that Phe-Lys was cleaved most rapidly with a half-life of 8 min, followed closely by Val-Lys with a half-life of 9 min. In stark contrast, Val-Cit showed a half-life of 240 min. They also found that removal of the PAB group reduced the cleavage rate, presumably through steric interference with enzyme binding. Another study compared the potency of auristatin derivative MMAE linked by dipeptide linkers Phe-Lys and Val-Cit and an analogous hydrazone linker. The Val-Cit linker proved to be over 100 times as stable as the hydrazone linker in human plasma. Most significantly, the Phe-Lys linker was substantially less stable than Val-Cit in human plasma, which accounts for its current popularity (see, e.g., Doronina et al. (2003) Nat. Biotechnol. 21(7): 778-784).

[0180] Other peptide linkers include, but not limited to, alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, GGGG, PSGSP, PSPSP, KKKK, RRRR, ASASA, GGSGGS, GGGGS, GGGGS GGGGS, GGGGS GGGGS GGGGS, GGGGS GGGGS GGGGS GGGGS, GGGGS GGGGS GGGGS GGGGSGGGGS, GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS, GGGGS GGGGS GGGGS FK GGGGS GGGGS GGGGS, and GGGGS GGGGS GGGGS VA GGGGS GGGGS GGGGS.

[0181] In some cases, the linkers described herein utilize PASYLATION®-technology, which is a genetic fusion with conformationally disordered polypeptide sequences composed of the amino acids Pro, Ala, and / or Ser. In other cases, the linkers described herein are selected from the ones disclosed in patents such as U.S. Patent 10,961,287, which is incorporated by reference herein in its entirety.

[0182] In some cases, the linkers described herein are non-peptide linkers. A glucuronide linker incorporates a hydrophilic sugar group that is cleaved by the lysosomal enzyme beta glucuronidase. Once the sugar is cleaved from the phenolic backbone, self-immolation of the PAB group releases the conjugated moiety (see, e.g., Jeffrey et al. (2006) Bioconjug. Chem. 17(3): 831-840). In some cases, the linkers described herein are derived from natural multidomain proteins (see, e.g., Chen at al., (2013) Adv Drug Deliv Rev. 65(10): 1357-1369).

[0183] The targeting moieties disclosed herein may be attached to the drug molecules disclosed herein via certain functional groups or via certain reactions. In some cases, the linker disclosed herein comprises a functional group that is reactive with a corresponding functional group on the targeting moieties disclosed herein and / or the drug molecules disclosed herein. A bifunctional linker has one functional group reactive with a group on the targeting moieties disclosed herein and another functional group reactive on the drug molecules disclosed herein and can be used to form the desired conjugate. In some instances, a heterobifunctional linker comprises two or more different reactive groups that react with sites on the targeting moieties disclosed herein and the drug molecules disclosed herein, respectively. For example, a heterobifunctional crosslinker such as cysteine may comprise an amine reactive group and a thiol-reactive group can interact with an aldehyde on a derivatized peptide. Additional combinations of reactive groups suitable for heterobifunctional crosslinkers include, for example, amine- and sulfhydryl reactive groups; carbonyl and sulfhydryl reactive groups; amine and photoreactive groups; sulfhydryl and photoreactive groups; carbonyl and photoreactive groups; carboxylate and photoreactive groups; and arginine and photoreactive groups. Such reactions and functional groups are illustrative and non-limiting. Other illustrative suitable reactive groups include, but are not limited to thiol ( — SH), carboxylate (COOH), carboxyl ( — COOH), carbonyl, amine (NH2), hydroxyl ( — OH), aldehyde ( — CHO), alcohol (ROH), ketone (R2CO), active hydrogen, ester, sulfhydryl (SH), phosphate ( — PO3), or photoreactive moieties. Amine reactive groups include, but are not limited to e.g.,isothiocyanates, isocyanates, acyl azides, NHS esters, sulfonyl chlorides, aldehydes and glyoxals, epoxides and oxiranes, carbonates, arylating agents, imidoesters, carbodiimides, and anhydrides. Thiol-reactive groups include, but are not limited to e.g., haloacetyl and alkyl halide derivates, maleimides, aziridines, acryloyl derivatives, arylating agents, and thiol-disulfides exchange reagents. Carboxylate reactive groups include, but are not limited to e.g., diazoalkanes and diazoacetyl compounds, such as carbonyldiimidazoles and carbodiimides. Hydroxyl reactive groups include, but are not limited to e.g., epoxides and oxiranes, carbonyldiimidazole, oxidation with periodate, N,N'-disuccinimidyl carbonate or N- hydroxylsuccimidyl chloroformate, enzymatic oxidation, alkyl halogens, and isocyanates. Aldehyde and ketone reactive groups include, but are not limited to e.g., hydrazine derivatives for schiff base formation or reduction amination. Active hydrogen reactive groups include, but are not limited to e.g., diazonium derivatives for mannich condensation and iodination reactions. Photoreactive groups include, but are not limited to e.g., aryl azides and halogenated aryl azides, benzophenones, diazo compounds, and diazirine derivatives.

[0184] In some instances, the linkers described herein connect the targeting moiety to the drug molecule by a cycloaddition reaction between an azide and an alkyne to form a triazole, wherein the azide and the alkyne may be located on the targeting moiety, the drug molecule, or the linker. In some instances, an alkyne may be a cyclic alkyne, e.g., a cyclooctyne. In some instances, an alkyne may be bicyclononyne (also known as bicyclononyne or BCN) or substituted bicyclononyne. In some instances, a cyclooctane is as described in International Patent Application Publication WO2011136645, published on Nov. 3, 2011, entitled, “Fused Cyclooctyne Compounds And Their Use In Metal free Click Reactions”. In some instances, an azide may be a sugar or carbohydrate molecule that comprises an azide. In some instances, an azide may be 6-azido-6-deoxygalactose or 6-azido-N-acetylgalactosamine. In some instances, a sugar or carbohydrate molecule that comprises an azide is as described in International Patent Application Publication W02016170186, published on Oct. 27, 2016, entitled, “Process For The Modification Of A Glycoprotein Using A Glycosyltransferase That Is Or Is Derived From A P(l,4)-N-Acetylgalactosaminyltransferase”. In some instances, a cycloaddition reaction between an azide and an alkyne to form a triazole, wherein the azide and the alkyne may be located on the targeting moiety, drug molecule, or the linker is as described in International Patent Application Publication WO2014065661, published on May 1, 2014, entitled, “Modified antibody, antibody-conjugate and process for the preparation thereof’; or International Patent Application Publication W02016170186, published on Oct. 27, 2016, entitled, “Process ForThe Modification Of A Glycoprotein Using A Glycosyltransferase That Is Or Is Derived From A P(1 ,4)-N-Acetylgalactosarninyltransferase”.

[0185] In some instances, the linkers described herein connect to the targeting moiety to the drug molecule by the Diels-Alder reaction between a dienophile and a diene / hetero-diene, wherein the dienophile and the diene / hetero-diene may be located on the targeting moiety, the drug molecule, or the linker. In some instances a linker is connected to the targeting moiety and / or the drug molecule by other pericyclic reactions, e.g. ene reaction. In some instances, the linker disclosed herein is connected to the targeting moiety and / or the drug molecule by an amide, thioamide, or sulfonamide bond reaction. In some instances, the linker disclosed herein connect to the targeting moiety to the drug molecule by a condensation reaction to form an oxime, hydrazone, or semicarbazide group existing between the linker, the targeting moiety, and the drug molecule.

[0186] In some instances, the linker disclosed herein connects the targeting moiety to the drug molecule by a conjugate addition reactions (also herein referred to as 'binding agent', ' bioconjugate' or 'bioconjugation') between a nucleophile, e.g. an amine or a hydroxyl group, and an electrophile, e.g. a carboxylic acid or an aldehyde. In some instances, a nucleophile may exist on a linker and an electrophile may exist on the targeting moiety or the drug molecule prior to a reaction between a linker and the targeting moiety or the drug molecule. In some instances, an electrophile may exist on a linker and a nucleophile may exist on the targeting moiety or the drug molecule prior to a reaction between a linker and the targeting moiety or the drug molecule. In some instances, an electrophile may be an azide, a silicon centers, a carbonyl, a carboxylic acid, an anhydride, an isocyanate, a thioisocyanate, a succinimidyl ester, a sulfosuccinimidyl ester, a maleimide, an alkyl halide, an alkyl pseudohalide, an epoxide, an episulfide, an aziridine, an aryl, an activated phosphorus center, and / or an activated sulfur center. In some instances, a nucleophile may be an optionally substituted alkene, an optionally substituted alkyne, an optionally substituted aryl, an optionally substituted heterocyclyl, a hydroxyl group, an amino group, an alkylamino group, an anilido group, or a thiol group.

[0187] In some instances, the linkers described herein further comprises a spacer, e.g., a polyethylene glycol spacer or an acyl / carbomoyl sulfamide spacer, e.g., a HydraSpace™ spacer. In some instances, a spacer is as described in Verkade, J. M. M. et al., “A Polar Sulfamide Spacer Significantly Enhances the Manufacturability, Stability, and Therapeutic Index of Antibody-Drug Conjugates”, Antibodies, 2018, 7, 12, which is incorporated by reference herein in its entirety.

[0188] Generally the linkers disclosed herein have no specific biological activity other than to join the proteins or to preserve some minimum distance or other spatial relationship between them. However, the constituent amino acids of the spacer may be selected to influence some property of the molecule such as the folding, net charge, or hydrophobicity. In certain instances the linker may comprise an enzymatic cleavage site.

[0189] After chemical synthesis, biological expression, or purification, the fusion protein may possess a conformation substantially different than the native conformations of the constituent polypeptides. In this case, it may be necessary to denature and reduce the polypeptide and then to cause the polypeptide to re-fold into the preferred conformation. Methods of reducing and denaturing proteins and inducing re-folding are well known to those of skill in the art (See, Debinski et al. (1993) J. Biol. Chem., 268: 14065-14070; Kreitman and Pastan (1993) Bioconjug. Chem., 4: 581-585; and Buchner, et al. (1992) Anal. Biochem., 205: 263-270).

[0190] In some instances, one of the targeting moiety and drug molecule is a peptide / protein or an antibody or an antigen binding fragment thereof and the other is a nucleotide. In some cases, the peptide / protein or an antibody or an antigen binding fragment thereof comprises a nonnatural amino acid to which the nucleotides can be covalently linked. In some cases, the peptide / protein or an antibody or an antigen binding fragment thereof is covalently linked to the nucleotide via conjugation to a lysine residue or a cysteine residue of the peptide / protein or an antibody or an antigen binding fragment thereof. In some cases, the nucleotide is conjugated to a cysteine residue of the peptide / protein or an antibody or an antigen binding fragment thereof via a maleimide-containing linker, optionally wherein the maleimide-containing linker comprises a maleimidocaproyl or maleimidomethyl cyclohexane- 1 -carboxylate group. In some instances, peptide / protein or an antibody or an antigen binding fragment thereof is glycosylated that comprises at least one sugar moiety to which the nucleotide is covalently linked. In some cases, the at least one sugar moiety comprises at least one sugar moiety that is a branched mannose. In some cases, the peptide / protein or an antibody or an antigen binding fragment thereof is glycosylated that comprises more than one sugar moieties, and each of all or a portion of the more than one sugar moieties is covalently linked to a separate nucleotide. In some cases, the peptide / protein or an antibody or an antigen binding fragment thereof is a fully-glycosylated or a partially-glycosylated. A partially-glycosylation may be produced via chemical or enzymatic means. In some cases, a partially-glycosylation is produced in a cell that is deficient for an enzyme in the N- or O-glycosylation pathway. In some instances, the linkers described herein are connected to the peptide / protein or an antibody or an antigen binding fragmentthereof via a phosphate, thioether, ether, carbon-carbon, or amide bond. In some instances, the linkers described herein are connected to the nucleotide through a phosphate or phosphorothioate group, e.g. a terminal phosphate of an oligonucleotide backbone.

[0191] In some instances, both the targeting moiety and drug molecule are peptides / proteins or antibodies or antigen binding fragments thereof. Accordingly, in some cases, the targeting moiety and drug molecule can be expressed as a fusion protein. In some specific cases, the targeting moiety and drug molecule are directly attached to each other. In some specific cases, the targeting moiety and drug molecule are connected via an intervening amino acid. In some specific cases, the targeting moiety and drug molecule are connected through a peptide linker. Specifically, the linkers can be joined to the constituent amino acids through their side groups (e.g., through a disulfide linkage to cysteine), while in other instances, the linkers are joined to the alpha carbon amino and carboxyl groups of the terminal amino acids when such are present.

[0192] For generating the fusion protein, generally this involves creating a DNA sequence that encodes the fusion protein, placing the DNA in an expression cassette under the control of a particular promoter, expressing the protein in a host, isolating the expressed protein and, if required, renaturing the protein. DNA encoding the fusion proteins can be prepared by any suitable method, including, for example, cloning and restriction of appropriate sequences or direct chemical synthesis by methods such as the phosphotriester method of Narang et al. (1979) Meth. Enzymol. 68: 90-99; the phosphodiester method of Brown et al. (1979) Meth. Enzymol. 68: 109-151; the diethylphosphoramidite method of Beaucage et al. (1981) Tetra. Lett., 22: 1859-1862; and the solid support method ofU.S. Pat. No. 4,458,066. In certain instances, DNA encoding fusion proteins constructs described herein may be cloned using DNA amplification methods such as polymerase chain reaction (PCR). Thus, for example, the nucleic acid sequence encoding the targeting moieties disclosed herein can be PCR amplified, using primers containing the designed restriction sites. This produces a nucleic acid encoding the targeting moieties disclosed herein and having terminal restriction sites. Similarly the nucleic acid encoding the drug molecules disclosed herein can be provided having complementary restriction sites. Ligation of sequences and insertion into a vector produces a vector encoding the fusion protein. The nucleic acid sequences encoding the fusion proteins can be expressed in a variety of host cells, including E. coli, other bacterial hosts, yeast, and various higher eukaryotic cells such as the COS, CHO and HeLa cells lines and myeloma cell lines. The recombinant protein gene will be operably linked to appropriate expression control sequences for each host. For E. coli this includes a promoter such as the T7, trp, or lambda promoters, aribosome binding site and preferably a transcription termination signal. For eukaryotic cells, the control sequences will include a promoter and preferably an enhancer derived from immunoglobulin genes, SV40, cytomegalovirus, etc., and a polyadenylation sequence, and may include splice donor and acceptor sequences. The plasmids can be transferred into the chosen host cell by well-known methods such as calcium chloride transformation for E. coli and calcium phosphate treatment or electroporation for mammalian cells. Cells transformed by the plasmids can be selected by resistance to antibiotics conferred by genes contained on the plasmids, such as the amp, gpt, neo and hyg genes.

[0193] Once expressed, the recombinant fusion proteins can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like (see, generally, R. Scopes (1982) Protein Purification, Springer- Verlag, N.Y.; Deutscher (1990) Methods in Enzymology Vol. 182: Guide to Protein Purification., Academic Press, Inc. N.Y.). Substantially pure compositions of at least about 90 to 95% homogeneity are preferred, and 98 to 99% or more homogeneity are most preferred for pharmaceutical uses. Once purified, partially or to homogeneity as desired, the polypeptides may then be used therapeutically.

[0194] In some cases, certain modifications can be made to the fusion proteins without diminishing their biological activity. Some modifications may be made to facilitate the cloning, expression, or incorporation of the drug molecules or targeting moieties disclosed herein into a fusion protein. Such modifications include, for example, a methionine added at the amino terminus to provide an initiation site, or additional amino acids placed on either terminus to create conveniently located restriction sites or termination codons.

[0195] In some instances, one of the targeting moiety and drug molecule is a peptide / protein or an antibody or an antigen binding fragment thereof and the other is a small molecule. The exemplary linkers can be found in U.S. Patents 10864279, 9814784, 11173214, 11104968, 9872924, 10537644, U.S. patent applications US20180169262, US20220072137, and US20180311375, which are incorporated by reference herein in their entireties.

[0196] In some instances, the drug conjugate is an AOC (antibody oligonucleotide conjugate) comprising a fatty acid conjugated oligonucleotide. In this embodiment the targeting moiety is a fatty acid (or a fatty acid ligand) and the drug molecule is an oligonucleotide (e.g. siRNA). Such a drug conjugate is useful for treating neuroscience, neuromuscular and CNS indications e.g., neuromuscular disorder Charcot-Marie-Tooth (CMT) disease, such as CMT type 1A (CMT1A), axonal loss, dysfunction of the neuronal axon, or abnormal axon-Schwann cellinteraction. The usage of fatty acid receptors conjugate enable cellular uptake and fatty acid albumin interactions to promote biodistribution of RNA therapeutics beyond the liver (extrahepatic delivery). For example, it decreases the expression of PMP22 protein in Schwann cells, which is the target cell type for the development, maintenance, and function of peripheral nerves. The fatty acid conjugated oligonucleotide facilitates the targeted approach of the present invention by conjugating siRNAs to naturally occurring fatty acids to improve the biodistribution and cellular uptake to tissues and cell types of interest.Other Properties of Drug Conjugates and Pharmaceutical Compositions

[0197] In certain instances, multiple targeting moieties disclosed herein can be attached to a single drug molecule disclosed herein. In certain instances, multiple the drug molecules disclosed herein can be attached to a single targeting moiety disclosed herein. In certain instances a single targeting moiety disclosed herein is attached to a single drug molecule disclosed herein. In some cases, the Drug molecule to the Targeting Moiety Ratio (DTMR) is 1 to 10 or any ratio between 2 to 10. In some embodiments, the Drug-Targeting Moiety Ratio (DTMR) as measured according to Matsuda, Y., Mendelsohn, BA., Chem Pharm Bull (Tokyo). 2021 ;69, 976, is between 2 to 8.

[0198] In some aspect, provided herein are pharmaceutical compositions comprising drug conjugates disclosed herein and pharmaceutically acceptable excipients. In some instances, the pharmaceutical compositions are formulated for parenteral, intravenous, subcutaneous, intrathecal, or intrasciatic injection.

[0199] In some instances, the drug conjugate provided herein preferably internalizes to Schwann cells. In some instances, the drug conjugate provided herein internalizes to Schwann cells at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 fold(s) more than it internalizes to a cell type other than Schwann cells.

[0200] In some instances, the drug conjugate provided herein enters blood stream and stays intact. In some instances, the drug conjugate provided herein enters blood stream and effectively crosses the blood nerve barrier. In some instances, the drug conjugate provided herein enters blood stream, effectively crosses the blood nerve barrier, and enriches in sciatic nerves.

[0201] The pharmaceutical composition described herein can be prepared to include the drug conjugates disclosed herein, in a form suitable for administration to a subject using carriers, excipients, and vehicles. Exemplary excipients include magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talc, milk protein, gelatin, starch, vitamins,cellulose and its derivatives, animal and vegetable oils, polyethylene glycols and solvents, such as sterile water, alcohols, glycerol, and polyhydric alcohols. Intravenous vehicles include fluid and nutrient replenishers. Preservatives include antimicrobial, anti-oxidants, chelating agents, and inert gases. Other pharmaceutically acceptable vehicles include aqueous solutions, nontoxic excipients, including salts, preservatives, buffers and the like, as described, for instance, in Remington: The Science and Practice of Pharmacy, 21st Ed., Gennaro, Ed., Lippencott Williams & Wilkins (2005), and The United States Pharmacopeia: The National Formulary (USP 36 NF31), published in 2013. The pH and exact concentration of the various components of the pharmaceutical composition are adjusted according to routine skills in the art. See Goodman and Gilman's, The Pharmacological Basis for Therapeutics.

[0202] The pharmaceutical compositions described herein may be administered locally or systemically. The therapeutically effective amounts will vary according to factors, such as the degree of infection in a subject, the age, sex, and weight of the individual. Dosage regimes can be adjusted to provide the optimum therapeutic response. For example, several divided doses can be administered daily or the dose can be proportionally reduced as indicated by the exigencies of the therapeutic situation.

[0203] The pharmaceutical composition can be administered in a convenient manner, such as by injection (e.g., subcutaneous, intravenous, intraorbital, intrasciatic, intrathecal, and the like), oral administration, ophthalmic application, inhalation, topical application, or rectal administration. Depending on the route of administration, the pharmaceutical composition can be coated with a material to protect the pharmaceutical composition from the action of enzymes, acids, and other natural conditions that may inactivate the pharmaceutical composition. The pharmaceutical composition can also be administered parenterally or intraperitoneally. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.

[0204] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In some instances, the composition will typically be sterile and fluid to the extent that easy syringability exists. In some instances, the composition will be stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms, such as bacteria and fungi. The vehicle can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size, in the case of dispersion, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, isotonic agents, for example, sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride are used in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

[0205] Sterile injectable solutions can be prepared by incorporating the pharmaceutical composition in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the pharmaceutical composition into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.

[0206] In some cases, the pharmaceutical composition is formulated in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein, refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of pharmaceutical composition is calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle. The specification for the dosage unit forms are related to the characteristics of the pharmaceutical composition and the particular therapeutic effect to be achieve. The principal pharmaceutical composition is compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable vehicle in an acceptable dosage unit. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the ingredients.

[0207] The pharmaceutical composition can be orally administered, for example, in a carrier, e.g., in an enteric-coated unit dosage form. The pharmaceutical composition and other ingredients can also be enclosed in a hard or soft-shell gelatin capsule or compressed into tablets. For oral therapeutic administration, the pharmaceutical composition can be incorporated with excipients and used in the form of ingestible tablets, troches, capsules, pills, wafers, and the like. The percentage of the compositions and preparations can, of course, bevaried and can conveniently be between about 5% to about 80% of the weight of the unit. The tablets, troches, pills, capsules, and the like can also contain the following: a binder, such as gum tragacanth, acacia, corn starch, or gelatin; excipients such as dicalcium phosphate; a disintegrating agent, such as corn starch, potato starch, alginic acid, and the like; a lubricant, such as magnesium stearate; and a sweetening agent, such as sucrose, lactose or saccharin, or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring. When the dosage unit form is a capsule, it can contain, in addition to materials of the above type, a liquid carrier. Various other materials can be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules can be coated with shellac, sugar, or both. A syrup or elixir can contain the agent, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye, and flavoring, such as cherry or orange flavor. Any material used in preparing any dosage unit form should be of pharmaceutically acceptable purity and substantially non-toxic in the amounts employed. In addition, the pharmaceutical composition can be incorporated into sustained-release preparations and formulations.

[0208] For the polynucleotide molecule described herein, suitable pharmaceutically acceptable salts include (i) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine, etc.; (ii) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, and the like; and (iii) salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like.In some instances, the drug molecule disclosed herein is a protein degrader molecule. In this case, the protein degrader molecule may be conjugated to a targeting moiety for targeted delivery of the protein degrader molecule, achieving targeted and regulated protein degradation of the target molecule (Protein of Interest) such as PROTAC technology (see Mancarella, C., et al., Int J Mol Sci., 2023, 24, 16346, incorporated herein by reference in its entirety) or (ubiquitin-or ubiquitin-like proteasome machinery).Methods

[0209] In some aspects, provided herein are methods of targeted delivery of drugs to Schwann cells, by contacting the drug conjugates disclosed herein or the pharmaceutical compositions comprising the drug conjugates disclosed herein to the Schwann cells. In some instances, thedrug conjugates are internalized into the Schwann cells upon binding of the targeting moieties to the cell surface molecule expressed on the Schwann cells. In some instances, the cell surface molecule expressed on the Schwann cells is a cell surface receptor having an extracellular domain. In some instances, the cell surface molecules are enriched on the Schwann cell surface. In some instances, the drug conjugates are delivered to the Schwann cells by systemic delivery of the drug conjugates disclosed herein or the pharmaceutical compositions disclosed herein to the body of a subject. In some instances, the drug conjugates disclosed herein can penetrate the blood nerve barrier. In some instances, the drug conjugates disclosed herein can penetrate through the blood nerve barrier and can be enriched in sciatic nerves. In some aspects, provided herein are methods of preferable delivery of drugs to or into the Schwann cells, by contacting the drug conjugates disclosed herein or the pharmaceutical compositions comprising the drugs disclosed herein to the Schwann cells. In some instances, the drug conjugates are preferably internalized into the Schwann cells upon binding of the targeting moieties to the cell surface molecule expressed on the Schwann cells. In some instances, the cell surface molecule preferably expressed on the Schwann cells is a cell surface receptor having an extracellular domain. In some instances, the cell surface molecules are preferably expressed on the Schwann cell surface. In some instances, the cell surface molecules are expressed at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 2, 3, 4, 5 folds more in Schwann cells than other types of cells. In some instances, the methods provided herein ensure that the drugs are preferably internalized into the Schwann cells at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 2, 3, 4, 5, 6, 7, 8, 9, or 10 fold(s) more than being internalized into a cell type other than Schwann cells. In some instances, the drug conjugates are delivered to the Schwann cells by systemic delivery of the drug conjugates disclosed herein or the pharmaceutical compositions disclosed herein to the body of a subject. In some instances, the drug conjugates disclosed herein can penetrate the blood nerve barrier. In some instances, the drug conjugates disclosed herein can penetrate through the blood nerve barrier and can be enriched in sciatic nerves.

[0210] In some instances, the methods of targeted delivery disclosed herein possess improved pharmacokinetics. In specific instances, the methods of targeted delivery disclosed herein result in a higher Cmax (i.e., maximum concentration of the drug molecule in Schwann cells). In specific instances, the methods of targeted delivery disclosed herein result in a shorter Tmax when Cmax is reached in Schwann cells. In specific instances, the methods of targeted delivery disclosed herein result in a higher area under the concentration-time curve. In specificinstances, the methods of targeted delivery disclosed herein result in a longer ti / 2 (i.e., the time taken for the concentration of the drug molecule to fall by one half once distribution equilibrium has been reached in Schwann cells). Accordingly, in some cases, the methods of targeted delivery disclosed herein result in the circulation and availability of the drug conjugate in the subject for at least 1, 5, 10, 15, 20, 25, 30, 35, or 40 days. In some cases, the methods of targeted delivery disclosed herein result in the circulation and availability of the drug conjugate in the subject for about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120 days. In some cases, the methods of targeted delivery disclosed herein result in the circulation and availability of the drug conjugate in the subject for at most 90, 120, 150, 180, 210, or 240 days.

[0211] In some instances, the methods of targeted delivery disclosed herein result in reduced distribution in any other tissues, organs, or cell types than Schwann cells. In some instances, the methods of targeted delivery disclosed herein result in an insignificant amount or a minimum amount in any other tissues, organs, or cell types than Schwann cells. In some instances, the methods of targeted delivery disclosed herein are non-immunogenic. In some instances, the methods of targeted delivery disclosed herein are biodegradable. In some instances, the methods of targeted delivery disclosed herein are applicable for a varieties of different drug molecules.

[0212] In some instances, the methods of targeted delivery are achieved by parenteral, intravenous, subcutaneous, intrathecal, or intrasciatic injection of the pharmaceutical compositions disclosed herein. In some instances, the methods of targeted delivery are achieved by intravenous injection of the pharmaceutical compositions disclosed herein. In some instances, the methods of targeted delivery are achieved by topical delivery (see, e.g., Pitiot et al. (2022) Antibodies (Basel) 11(3):56.)

[0213] In another aspect, provided herein are methods of treating or preventing a peripheral demyelinating disease or a peripheral neuropathy, or alleviating or reducing symptoms of the peripheral demyelinating disease or the peripheral neuropathy in a subject in need thereof, comprising: providing the drug conjugates disclosed herein or the pharmaceutical compositions disclosed herein; and administering the subject the drug conjugates or the pharmaceutical compositions to treat the peripheral demyelinating disease or the peripheral neuropathy, to alleviate or reduce symptoms of the peripheral demyelinating disease or the peripheral neuropathy.

[0214] In some instances, the peripheral demyelinating disease or the peripheral neuropathy is Charcot-Marie-Tooth disorder, Guillain-Barre syndrome (acute inflammatory demyelinatingpolyradiculopathy type), schwannomatosis, chronic inflammatory demyelinating polyneuropathy, nerve trauma, post-chemotherapy neuropathy, diabetic peripheral neuropathy, migraine, Schwannomas, neurofibromatosis type 1 (NF1), malignant peripheral nerve sheath tumors (MPNSTs), endometriosis abdominopelvic pain or nerve injury.

[0215] In some instances where an elevation of the disease-associated molecule leads to the disease, the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to reduce expression of the disease-associated molecule by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some instances where an elevation of the disease-associated molecule leads to the disease, the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to reduce expression of the disease-associated molecule by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some instances where an elevation of the disease-associated molecule leads to the disease, the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to reduce activity of the disease-associated molecule by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some instances where an elevation of the disease-associated molecule leads to the disease, the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to reduce activity of the disease-associated molecule by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

[0216] In some instances, the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to reduce the expression of PMP22 by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some instances, the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to reduce the expression of PMP22 by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some instances, the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to reduce the activity of PMP22 by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some instances, the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to reduce the activity of PMP22 by about 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

[0217] In some instances, the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to reduce the expression of antiganglioside antibodies by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some instances, the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to reduce the expression of antiganglioside antibodies by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some instances, the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to reduce the activity of antiganglioside antibodies by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some instances, the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to reduce the activity of antiganglioside antibodies by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

[0218] In some instances where a lack of the disease-associated molecule leads to the disease, the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to increase expression of the disease-associated molecule by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some instances where a lack of the disease-associated molecule leads to the disease, the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to increase expression of the disease-associated molecule by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some instances where a lack of the disease-associated molecule leads to the disease, the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to increase activity of the disease-associated molecule by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some instances where a lack of the disease-associated molecule leads to the disease, the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to increase activity of the disease-associated molecule by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

[0219] In some instances, the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to increase the expression of the subunit ofSWI / SNF protein complexes encoded by gene SMARCB1 by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some instances, the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to increase the expression of the subunit of SWI / SNF protein complexes encoded by gene SMARCB1 by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some instances, the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to reduce the activity of the subunit of SWI / SNF protein complexes encoded by gene SMARCB1 by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some instances, the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to reduce the activity of the subunit of SWI / SNF protein complexes encoded by gene SMARCB1 by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

[0220] In some instances, the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to increase the expression of LZTR1 protein encoded by gene LZTR1 by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some instances, the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to increase the expression ofLZTRl protein encoded by gene LZTR1 by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some instances, the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to reduce the activity ofLZTRl protein encoded by gene LZTR1 by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some instances, the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to reduce the activity ofLZTRl protein encoded by gene LZTR1 by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

[0221] In some instances, the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to increase expression of a marker gene of myelination by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. Schwann cells exist in different developmental stages or wound repair phases, which express different Schwann cell-specific markers. Accordingly, in specific instances, the drug conjugate or the pharmaceutical composition isadministered in a dose and schedule effective to increase expression of a marker gene of Schwann cell precursors by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In specific instances, the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to increase expression of a marker gene of immature Schwann cell by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In specific instances, the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to increase expression of a marker gene of myelinating Schwann cell by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In specific instances, the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to increase expression of a marker gene of non-myelinating Schwann cell by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some cases, the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to increase expression of one or more the markers, including but are not limited to, SI 00, p75NTR, SoxlO, Sox2, GAP43, NCAM, Krox20, Oct6, MBP, and MPZ, at least by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

[0222] In another aspect, provided herein are methods of treating or preventing Neurofibroma and associated indications, disorders or types (i.e. localized, diffuse, and plexiform) in a subject in need thereof, comprising: providing the drug conjugates disclosed herein or the pharmaceutical compositions disclosed herein; and administering to the subject the drug conjugates or the pharmaceutical compositions to treat the Neurofibroma and associated indications, disorders or types, to alleviate or reduce symptoms of the Neurofibroma and / or associated indications, disorders or types. In some instances, the Neurofibroma disease- associated molecule, effected or modified (in terms of expression and / or activity) by the drug conjugate or pharmaceutical composition of the present invention, may be selected from receptor tyrosine kinases (RTKs), for example, of the Epidermal growth factor receptor family B (ErbB), such as EGFR, ErbB2, and ErbB3; additional RTKs include platelet-derived growth factor (PDGF), fibroblasts growth factor (FGF), insulin-like growth factor- 1 and vascular endothelial growth factor (VEGF). Nonreceptor protein tyrosine kinases (nrPTKs), include Src, BCR-ABL, and the JAK / STAT pathway. In some aspects, a drug conjugate or a pharmaceutical composition as disclosed herein, comprises SMW compound as a drug molecule inhibitingkinase domain of RTKs or nrPTKs (e.g. exemplified above) as potential therapeutic pathway to neurofibromas.

[0223] In some instances where an elevation of the disease-associated molecule leads to the disease, the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to reduce expression of the disease-associated molecule by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some instances where an elevation of the disease-associated molecule leads to the disease, the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to reduce expression of the disease-associated molecule by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some instances where an elevation of the disease-associated molecule leads to the disease, the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to reduce activity of the disease-associated molecule by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some instances where an elevation of the disease-associated molecule leads to the disease, the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to reduce activity of the disease-associated molecule by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

[0224] In some instances, the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to reduce the expression of RTKs or nrPTKs (e.g. exemplified above) by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some instances, the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to reduce the expression of RTKs or nrPTKs (e.g. exemplified above) by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some instances, the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to reduce the activity of RTKs or nrPTKs (e.g. exemplified above) by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some instances, the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to reduce the activity of RTKs or nrPTKs (e.g. exemplified above) by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,

[0225] In some instances where a lack of the disease-associated molecule leads to the disease, the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to increase expression of the disease-associated molecule by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some instances where a lack of the disease-associated molecule leads to the disease, the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to increase expression of the disease-associated molecule by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some instances where a lack of the disease-associated molecule leads to the disease, the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to increase activity of the disease-associated molecule by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some instances where a lack of the disease-associated molecule leads to the disease, the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to increase activity of the disease-associated molecule by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

[0226] In some instances, the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to increase the expression of the subunit of RTKs or nrPTKs (e.g. exemplified above) by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some instances, the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to increase the expression of RTKs or nrPTKs (e.g. exemplified above) by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some instances, the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to increase the activity of RTKs or nrPTKs (e.g. exemplified above) by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some instances, the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to increase the activity of RTKs or nrPTKs (e.g. exemplified above) by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.

[0227] In some instances, the disease associated molecules to be targeted and modulated by the drug conjugate of the present invention are kinases involved in Schwannomas and neurofibromatosis (NF) including the Ras / Raf-'MEK / ERK and PI3K / Akt / niTORCl pathways(Brown, R., Curr Oncol Rep., 2023,12, 1409; and Tamura, R. et al., Int J Mol Sci., 2022, 23, 5462 are incorporated herein by their entirety).

[0228] In some aspects, provided herein are methods of treating or diagnosing a subject with a disease associated with dysfunction and / or degeneration of Schwann cells and / or axons, and axon related disorders, comprising administering to a subject in need of such a treatment or diagnosis an effective amount of the drug conjugate as disclosed herein or a pharmaceutical composition as disclosed herein, comprising the drug conjugate.

[0229] In some instances, the methods provided herein comprise steps of monitoring the progress of, or detecting the therapeutic effect of the drug conjugate or the pharmaceutical composition as disclosed herein.Certain Terminology

[0230] The term "drug conjugate" as used herein encompasses, in a non-limiting manner, Antibody Drug Conjugate (ADC), Antibody Oligonucleotide Conjugate (AOC), Degraderantibody conjugates (DAC) or any conjugation of a therapeutic entity to a delivery entity.

[0231] The term “disease-associated molecule,” as used herein, generally refers to a molecule that is directly or indirectly associated with the pathogenesis, progression, prolonging, or recurrence of a disease. In some cases, the abnormal expression level of the disease-associated molecule is associated with the pathogenesis, progression, prolonging, or recurrence of the disease. In some cases, the abnormal activity of the disease-associated molecule is associated with the pathogenesis, progression, prolonging, or recurrence of the disease.

[0232] The term "protein degraders" or "degraders" as used herein refers to small molecular weight compounds that use the cell’s natural system (ubiquitin-or ubiquitin-like proteasome machinery) for disposing of spent or misfolded proteins. These small molecules are designed with two active ends: one that binds to the protein of interest and the other that binds to a ubiquitin ligase (protein called E3). These bifunctional molecules force a contact between the ubiquitin and the protein, applying it to be degraded. Afterwords, the process is repeated, enabling quick depletion of the unwanted protein. Thus the degraders are tagging the undesirable protein for ubiquitination and subsequent breakdown into amino acids in the proteasome. In the context of the present invention, eliminating certain proteins e.g. by protein degraders is used for cancer indications. The protein degraders are herein used as a targeting moiety conjugated to a drug molecule for treating Schwann cell-related oncologic indications such as schwannoma or schwannomatosis.

[0233] Degrader-antibody conjugates (DACs) as used herein refer to entities that combine a proteolysis targeting chimera (PROTAC) or molecule payload with an antibody (e.g. monoclonal antibody), usually via a type of chemical linker.

[0234] The term “conservative amino acid substitution,” as used herein, generally refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art such as are found in references which compile such methods, e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Fourth Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2012, or Current Protocols in Molecular Biology, F. M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative substitutions of amino acids include substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

[0235] The term “antibody,” as used herein, generally refers to a polypeptide that includes at least one immunoglobulin variable domain or at least one antigenic determinant, e.g., paratope that specifically binds to an antigen. In some instances, an antibody is a full-length antibody. In some instances, an antibody is a chimeric antibody. In some instances, an antibody is a humanized antibody. However, in some instances, an antibody is a Fab fragment, a F(ab')2 fragment, a Fv fragment or an scFv fragment.

[0236] As used herein, Fab fragment refers to a region on an antibody that binds to antigens. It is composed of one constant and one variable domain of each of the heavy and the light chain. These domains shape the paratope, which is the antigen-binding site, at the amino terminal end of the monomer.

[0237] As used herein, Fc region refers to the tail region of an antibody that interacts with cell surface receptors called Fc receptors and some proteins of the complement system. This property allows antibodies to activate the immune system. In some instances, the Fc regions of immunoglobulins bear a highly conserved N-glycosylation site.

[0238] In some instances, an antibody is a nanobody derived from a camelid antibody or a nanobody derived from shark antibody. In some instances, an antibody is a diabody. In some instances, an antibody comprises a framework having a human germline sequence. In other instances, an antibody comprises a heavy chain constant domain selected from the group consisting of IgG, IgGl, IgG2, IgG2A, IgG2B, IgG2C, IgG3, IgG4, IgAl, IgA2, IgD, IgM, and IgE constant domains. In some instances, an antibody comprises a heavy (H) chain variableregion (abbreviated herein as VH), and / or a light (L) chain variable region (abbreviated herein as VL). In some instances, an antibody comprises a constant domain, e.g., an Fc region. An immunoglobulin constant domain refers to a heavy or light chain constant domain. Human IgG heavy chain and light chain constant domain amino acid sequences and their functional variations are known. With respect to the heavy chain, in some instances, the heavy chain of an antibody described herein can be an alpha (a), delta (A), epsilon (s), gamma (y) or mu (p) heavy chain. In some instances, the heavy chain of an antibody described herein can comprise a human alpha (a), delta (A), epsilon (s), gamma (y) or mu (p) heavy chain. In a particular instance, an antibody described herein comprises a human gamma 1 CHI, CH2, and / or CH3 domain. In some instances, the amino acid sequence of the VH domain comprises the amino acid sequence of a human gamma (y) heavy chain constant region, such as any known in the art. Non-limiting examples of human constant region sequences have been described in the art, e.g., see U.S. Pat. No. 5,693,780 and Kabat E A et al., (1991) supra. In some instances, the VH domain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or at least 99% identical to any of the variable chain constant regions provided herein. In some instances, an antibody is modified, e.g., modified via glycosylation, phosphorylation, sumoylation, and / or methylation. In some instances, an antibody is a glycosylated antibody, which is conjugated to one or more sugar or carbohydrate molecules. In some instances, the one or more sugar or carbohydrate molecule are conjugated to the antibody via N-glycosylation, O-glycosylation, C- glycosylation, glypiation (GPI anchor attachment), and / or phosphoglycosylation. In some instances, the one or more sugar or carbohydrate molecule are monosaccharides, disaccharides, oligosaccharides, or glycans. In some instances, the one or more sugar or carbohydrate molecule is a branched oligosaccharide or a branched glycan. In some instances, the one or more sugar or carbohydrate molecule includes a mannose unit, a glucose unit, an N-acetylglucosamine unit, an N-acetylgalactosamine unit, a galactose unit, a fucose unit, or a phospholipid unit. In some instances, an antibody is a construct that comprises a polypeptide comprising one or more antigen binding fragments of the disclosure linked to a linker polypeptide or an immunoglobulin constant domain. Linker polypeptides comprise two or more amino acid residues joined by peptide bonds and are used to link one or more antigen binding portions. Examples of linker polypeptides have been reported (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2: 1121-1123). Still further, an antibody may be part of a larger immunoadhesion molecule, formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples ofsuch immunoadhesion molecules include use of the streptavidin core region to make a tetrameric scFv molecule (Kipriyanov, S. M., et al. (1995) Human Antibodies and Hybridomas 6:93-101) and use of a cysteine residue, a marker peptide and a C-terminal polyhistidine tag to make bivalent and biotinylated scFv molecules (Kipriyanov, S. M., et al. (1994) Mol. Immunol. 31: 1047-1058).

[0239] The term “covalent,” as used herein, generally refers to a characteristic of two or more molecules being linked together via at least one covalent bond. In some instances, two molecules can be covalently linked together by a single bond, e.g., a disulfide bond or disulfide bridge, that serves as a linker between the molecules. However, in some instances, two or more molecules can be covalently linked together via a molecule that serves as a linker that joins the two or more molecules together through multiple covalent bonds. In some instances, a linker may be a cleavable linker. However, in some instances, a linker may be a non-cleavable linker.

[0240] The term “specifically binds,” as used herein, generally refers to the ability of a molecule to bind to a binding partner with a degree of affinity or avidity that enables the molecule to be used to distinguish the binding partner from an appropriate control in a binding assay or other binding context. With respect to an antibody, the term, “specifically binds”, refers to the ability of the antibody to bind to a specific antigen with a degree of affinity or avidity, compared with an appropriate reference antigen or antigens, that enables the antibody to be used to distinguish the specific antigen from others, e.g., to an extent that permits preferential targeting Schwann cells, through binding to the antigen, as described herein. In some instances, an antibody specifically binds to a target if the antibody has a KD for binding the target of at least about 10"4M, 10"5M, 10"6M, 10"7M, 10"8M, 10"9M, 10"10M, 10"11M, 10"12M, 10"13M, or less.

[0241] The term “(poly)peptide” or “protein” as used herein, are used interchangeably, and generally refers to a series of amino acid residues joined by peptide bonds (i.e., a polymer of amino acids) and include modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs. Illustrative polypeptides or proteins include gene products, naturally occurring proteins, homologs, paralogs, fragments and other equivalents, variants, and analogs of the above.

[0242] The terms “subject,” “individual,” “mammal”, and “patient,” as used herein, used interchangeably and generally refer to may be and refer to humans, as well as non-human mammals (e.g., non-human primates, canines, equines, felines, porcines, bovines, ungulates, lagomorphs, and the like). In various instances, the subject can be a human (e.g., adult male,adult female, adolescent male, adolescent female, male child, female child) under the care of a physician or other health worker in a hospital, as an outpatient, or other clinical context. In certain instances, the subject may not be under the care or prescription of a physician or other health worker.

[0243] The term “linker,” as used herein, generally refers to is a molecule that is used to join two or more molecules. In certain instances, the linker is capable of forming covalent bonds to both molecule(s). Suitable linkers include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. In further instances the linker is selected from a cleavable linker, a non-cleavable linker, covalently bound linker such as by chemical conjugation, linkers based on non-covalent interactions, a carbohydrate linker, a peptide linker, a glycan linker or a non-peptide linker.

[0244] Whenever the term “at least,” “greater than,” or “greater than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “at least,” “greater than” or “greater than or equal to” applies to each of the numerical values in that series of numerical values. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0245] The term “a,” “an” and “the” include plural referents unless the content clearly dictates otherwise.

[0246] The term “about,” particularly in reference to a given quantity, is meant to encompass deviations of plus or minus ten percent.

[0247] While various instances of the disclosure have been shown and described herein, it will be obvious to those skilled in the art that such instances are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the instances of the disclosure described herein may be employed.EXAMPLES

[0248] The following is a description of various methods and materials used in the studies, and are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present disclosure, and are not intended to limit the scope of what the inventors regard as their disclosure nor are they intended to represent that the experiments below were performed and are all of the experiments that may be performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, percentages, etc.), but some experimental errors and deviations should be accounted for.Example 1: anti-Gliomedin antibody expression and purificationMethodsRecombinant expression of mouse anti-Gliomedin antibody (Ab)

[0249] Reference is now made to the protein sequence of mouse anti-Gliomedin antibody of the following genetic constructs:

[0250] pcDNA3.4-mouse anti-Gliomedin-mlgGl (mouse Immunoglobulin Gl) long or heavy chain (VH): amino acid sequence as set forth in SEQ ID NO:1 (within the scope are sequences having at least 90%, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1) or a functional variant thereof.

[0251] pcDNA3.4-mouse anti-Gliomedin-mCk short or light chain (VL): amino acid sequence as set forth in SEQ ID NO: 2 (within the scope are sequences having at least 90%, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:2) or a functional variant thereof.

[0252] It is noted that the above mouse anti-Gliomedin antibody recognizes both mouse and human Gliomedin. It is within the scope of the present invention that a humanized antibody is prepared from each of the above non-human (mouse) species, whose protein sequences have been modified to increase their similarity to antibody variants produced naturally in humans. It is a common procedure that the process of "humanization" is applied to antibodies developed (in a non-human source) for administration to humans. In such cases, the complementary determining regions (CDRs) sequences from the mouse or other non-human antibodies are maintained in the human frameworks produced.

[0253] Reference is now made to the herein disclosed CDR sequences of anti-Gliomedin antibody (both for the Light and Heavy chains) used in the present invention to recognize and bind Gliomedin in Schwann cells. The CDR sequences are provided as Kabat and IMGT numbering schemes. It is herein acknowledged that the schemes are for the numbering of amino acid residues in antibodies based upon variable regions. The schemes are useful for accurate identification and subsequent comparison of variable regions of different antibodies. Non limiting examples of commonly used numbering schemes include IMGT, Kabat Chothia, Martin (Enhanced Chothia or AbM) and Honneger’s numbering scheme (AHo).

[0254] Reference is now made to Table 2 presenting the anti-Gliomedin CDRs sequence and their position in the Heavy Chain (VH) as set forth in SEQ ID NO: 1, according to Kabat and IMGT numbering schemes.Table 2: CDRs in the anti-Gliomedin Heavy Chain (VH) according to Kabat & IMGT numbering schemes

[0255] Reference is now made to Table 3 presenting the anti-Gliomedin CDRs sequence and their position in the Light Chain (VL) as set forth in SEQ ID NO: 2, according to Kabat and IMGT numbering schemes.Table 3: CDRs in the anti-Gliomedin Light Chain (VL) according to Kabat & IMGT numbering schemes

[0256] The CDR sequences SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 19, 20 and 30 were annotated using the abYsis database providing SEQ ID NOs: 1 or 2 as the input.

[0257] In one embodiment, the invention pertains to a polypeptide comprising the amino acid of any one of SEQ ID NOs: 3-22, and 30 or a fragment thereof. In another embodiment the invention pertains to a sequence or fragment thereof exhibiting at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% sequence identity to any one of SEQ ID NOs: 3-22, and 30.

[0258] Plasmids were used to transfect ExpiCHO (Chinese Hamster Ovary) which were grown and harvested from the conditioned medium by centrifugation and buffered by 1.5M glycine, 1.5M NaCl, 5mM EDTA (final concentration) and adjusted to pH8.5 with IN NaOH. Supernatant was filtrated with 0.22um and Loaded onto MabSelect SuRe / Protein-G / Capto L column pre-equilibrated with Buffer A (1.5M glycine, 1.5M NaCl, 5mM EDTA, 20mM Tris, pH8.5). Antibody was eluted with Buffer C (lOOmM sodium citrate, 300mM NaCl, pH3.0). Neutralized immediately with Buffer D (IM Arginine, 400mM Succinic acid, pH 9.0). Loadelution onto Superdex200 column pre-equilibrated with final buffer (lx PBS, pH7.4). Samples were Concentrated and filtrated through MILLEX-MP 0.22um. hPMP22 and hGliomedin Stable cell line generation

[0259] The protein sequence referenced by UNIPROT of both hPMP22 (Q01453) and hGliomedin (Q6ZMI3), as set forth in SEQ ID NO:23 and SEQ ID NO:24, respectively, were synthesized for vector construction. The vector was transiently transfected to HEK293 cells by X-tremeGENE HP DNA Transfection Reagent according to the product manual. Then commercial Ab was used to verify whether the transiently transfected HEK293 cells could express target protein correctly by FACS. The verified vector was used for lentivirus package. Host cells are infected with packaged lentivirus. After 2~3 times infection, commercial Ab was used to verify whether the host cells could express target protein correctly by FACS or Western blot. The optimal concentration of antibiotics is used for pool selection. After about 2 weeks, the stable pool is verified whether could express target protein by FACS or Western blot. Then the stable pool is used for single clone generation by cell sorting. Single clone cells are cultured, expanded and selected with high expression level of target protein by FACS or Western blot.Transient transfection

[0260] X-tremeGENE HP from Roche is used and the product manual is followed to conduct the experiment. Briefly, plasmid X-tremeGENE HP complexes are prepared first and then cells are added to each well with plasmid and X-tremeGENE HP. After mixing gently, cells are cultured for ~48 hours at 37°C in a CO2 incubator till ready for related gene expression assays. Lentivirus package and infection

[0261] 293 T cells are seeded in multiple T175 cell culture flasks for transfection with related plasmids using POLO3000. After incubation overnight and changing the medium after 24 hours, virus supernatant is collected at both 48 hours and 72 hours after transfection. After centrifugation, the virus supernatant is concentrated if necessary and stored at -80°C with titer determined.

[0262] For infection, host cells are plated in 6- well plate and virus supernatant with definite MOI are added. Cells are incubated at 37°C in a CO2 incubator until it is ready to assay for gene expression after 48-72 hours. Infection can be performed once, or multiple times, depending on the expression level detection.Pool selection

[0263] For pool selection, a portion of cells ~48 hours after infection are seeded into a 10cm dish using complete culture medium containing optimal concentration of antibiotics for poolselection about 2 weeks. The surviving pool cells are expanded for pool validation and the following pool cryopreservation.Single clone generation

[0264] After validation, if the characterization result is positive, the cells cultured with appropriate antibiotics concentration for about 2 weeks are collected for sorting. During sorting, single cell seeding model is selected and total 3 plates of cells in 96-well plates are seeded to try to achieve the density of 1 cell per well. Cells grow under antibiotic selection for 2~3 weeks until single clones can be identified in some of the wells. Cells growing up will be expanded into 24-well plate and then 6-well plate to get enough cells for screening by FACS or Western blot. Validation assay will be performed to confirm the positive characterization before cry opreservation.Detection cells expression level by FACS assays

[0265] The cells are harvested according to standard procedures. The cells are resuspended with FACS buffer and incubated with diluted primary antibody for desired time on ice. The cells are then washed 2-3 times with FACS buffer. Centrifugation is performed and the supernatant is discarded before and after each wash. The cells are incubated with diluted secondary antibody for desired time on ice in the dark. The cells are resuspended in FACS buffer for FACS analysis.Detection cells expression level by permeabilized FACS assays

[0266] The cells are harvested according to standard procedures. The cells are resuspended with FACS buffer and 4% Formaldehyde to finally 2% Formaldehyde and incubated for 20 min at room temperature (RT). The cells are washed 2-3 times with FACS buffer. Then the cells are resuspended in permeabilization buffer (0.2% TritonX-100) and incubated for 5 min, RT. The cells are washed 2-3 times with FACS buffer and incubated with diluted primary antibody for desired time on ice. The cells are washed 2-3 times with FACS buffer. Then the cells are centrifugated and the supernatant is discarded before and after each wash. The cells are incubated with diluted secondary antibody for desired time on ice in the dark. Finally, the cells are resuspended in FACS buffer for FACS analysis.ResultsAnti-Gliomedin Ab purification and validation

[0267] Anti-Gliomedin Ab was purified to homogeneity as was confirmed by SDS-PAGE, shown by Fig.lA, and SEC (Superdex200, 60ml, XK16 / 30) shown by Fig. IB.

[0268] The anti-Gliomedin Ab was evaluated for its binding and recognition of human Gliomedin (hGliomedin) by ELISA. As shown by Fig. 2, all 3 batches of Abs tested (MOUSEANTI-Gliomedin-1-3), showed good binding ability on hGliomedin-ECD-Fc recombinant protein (Sino biological, 15889-H01H) by ELISA.Validation of CHOK1 cells infected by human Gliomedin (hGLDN) and PMP22- lentivirus

[0269] The stable expression of human PMP22 (hPMP22) was validated using the primary antibody mouse anti-Gliomedin-1 (internal synthesis, Lot: #CP2024090301) lOOnM (see Fig. 3A), Rabbit anti-PMP22 antibody (Abeam, cat: #EPR23112-110, 1 :600 dilution (permeabilized FACS)) (see Fig. 3B) and mouse Isotype IgG as control. Secondary antibodies used were Alexa Fluor® 647 (goat anti-mlgG (H+L), Invitrogen, cat: #A21235, 1 :500 dilution) and Alexa Fluor® 647 (goat anti-rabbit (H+L), Invitrogen, cat: #A21244, 1:500 dilution).

[0270] As can be seen in Figs. 3A and 3B, the FACS analysis validates that CHOK1 cells infected by hGLDN and PMP22 lentivirus express human Gliomedin and PMP22 proteins.Example 2: Activity of siRNA on CHO-transfected hPMP22 / hGliomedinMethods siRNA synthesis

[0271] The following siRNA variants for reducing expression of hPMP22 (in Schwann cells), were prepared by standard synthesis, with and without the following modifications, or any other siRNA stabilization modification known in the art (see above):5' Modification: / 5'SMCC N / 3' modification: / 3'Phos / siRNA-1: UGGACACGCAACUGAUCUCUG- SEQ ID NO:25 siRNA-2: CUCGGAUUACUCCUACGGUUU- SEQ ID NO:26 siRNA-3: GCUGUUGAUUGAAGAUGUA- SEQ ID NO: 27 siRNA-4: CCUGAAUAAUUCUGUGUAA- SEQ ID NO:28 siRNA-5: AUACCAACUGUGUGGACUA- SEQ ID NO:29

[0272] In one embodiment, the invention encompasses a polynucleotide molecule comprising the nucleic acid sequence of any one of SEQ ID NOs: 25-29 or a fragment thereof, wherein the sequence or fragment exhibits at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% sequence identity to any one of SEQ ID NOs: 25-29.

[0273] All samples were prepared RNase free with HPLC (High-performance liquid chromatography) purification and delivered as dry powder.siRNA transfection

[0274] Cells were plated (50k / well) into 6-well plates and incubated overnight. A pool of RNAimax was made as follows: 18 pL of RNAimax +300 pL of opti-MEM for each condition. Two control siRNAs included (Scrambled and ON-TARGETplus SMARTpool human KIF11). siRNAs were prepared in Opti-MEM: 6 pL of 10 pM siRNA stock +300 pL of opti-MEM. The siRNA was combined with the diluted RNAimax and incubated at RT for 15 min. An amount of 150 pL / well mix was added per well. This leads to 1.5ml final volume and a 10 nM siRNA final RNA concentration in each well. The mixture was incubated overnight and was replaced with pre-warmed culture media. Viability (Beckman Vi-cell counter or CTG assay) was checked and 72h samples were collected.Quantitative Real-time PCRTotal RNA Extraction was performed, and the extracted RNA was quantified by NanoDrop2000. RT-PCR conducted, and Quantitative Real-time qPCR by TaqMan Gene Expression Assays were performed. The raw data were subjected to comparative CT method(ACT) and analysis.ResultsActivity of siRNA on CHO-transfected hPMP22 / hGliomedin

[0275] Cells were seeded into 6-well plates one day before transfection. On day 1 cells were transfected with siRNAs. On day 2, the cell medium was changed to full cell culture medium. At day 4, cells were collected from 6-well plates and hPMP22 mRNA levels were evaluated, normalized using qPCR Mouse ACTB Endogenous Control (ABI, 4352341E).

[0276] Reference is now made to Table 2 presenting the samples used in the above experiment.Table 2: Sample list

[0277] As can be seen in Fig. 4, all siRNA variants (SEQ ID Nos: 25-29) were able to reduce hPMP22 mRNA levels to approximately 40% as compared to control non-treated cells.Example 3: Construction of anti-Gliomedin- siRNA Antibody-Oliso-Conjusate (AOC)

[0278] A conjugate of mouse anti-Gliomedin antibody conjugation to siRNA is generated.

[0279] A purified anti-Gliomedin antibody (see Example 1), was reduced with TCEP to achieve appropriate reduction. The 5-SMCC-modified (succinimidyl 4-(N-maleimidomethyl) cyclohexane- 1 -carboxylate (SMCC)) siRNA variants 1-5 (comprising SEQ ID NO: 25-29) were mixed with the reduced anti-Gliomedin antibody at 25oC for >2 hours. Conjugation was analyzed by SEC-HPLC, SDS-PAGE, and HIC-HPLC to estimate DAR value, which is aimed to 2. The most optimal condition was selected to conjugate required quantity of antibody, characterize, purify, and final characterize.Example 4: Evaluation of targeted delivery of an Antibody- siRNA conjugate (AOC) in vitro assay

[0280] An Antibody Oligo Conjugate (AOC) comprising anti-Gliomedin antibody (e.g. see Example 1) conjugated to siRNA (e.g. see Example 2, SEQ ID NO: 25-29) (or anti-Gliomedin antibody conjugated to linker- siRNA) is incubated with CHO-transfected hPMP22 / hGliomedin cell lines. As a negative control, the cells are contacted with the isotype control mouse IgG conjugated to siRNA or anti-Gliomedin conjugated to scramble siRNA variant. Seeded modulated CHO cell are placed in a 96-well plate at a density of 5,000-10,000 cells per well and incubated with AOC variants (described above) in a humidified incubator at 37°C with 5% CO2 for in serial-diluted concentrations for 48 hours at the same conditions. Reduction of hPMP22 expression evaluated by FACS, RT-PCR and Western Blot, and 30-70% reduction was observed.Example 5: Evaluation of the Antibody- siRNA conjugate (AOCj functionality in C3-PMP mouse model

[0281] For in vivo analysis, C3-PMP mice (Also Known As: PMP22-C3; B6.Cg- Tg(PMP22)C3Fbas / J, The Jackson Laboratories) were intravenously injected with AntibodysiRNA-Conjugate (AOC) (as described above, e.g. Examples 1-3) or Antibody-scrambled- siRNA-Conjugate control variant. For validation of the AOCs silencing efficacy of a target or a disease-associated molecule (e.g. PMP22), injected mice were analyzed 6 weeks after once- weekly injection of about 5mg / kg dosing using immunohistochemistry, VGCN analysis, RT- qPCR, PMP22 quantification (e.g. Western blot), CatWalk Automated Gait Analysis, Grip Strength, Sciatic Nerve Conduction Studies and Sciatic Nerve Histology.

[0282] Example 6: Construction of anti-Gliomedin- Monomethyl auristatin E (MMAE) Antibody-Drug-Conjugate (ADC)

[0283] A conjugate of anti-Gliomedin antibody conjugation to MMAE is generated.

[0284] Nucleic acid sequence of anti-Gliomedin antibody is synthesized followed by plasmid construction in pCDNA3 and transfection into either in HEK293 and CHO cells for expression evaluation. In-vitro production was performed in 1000 ml roller bottle culture. Supernatants were collected and anti-Gliomedin was purified using MabSelect SuRe / Protein-G / Capto L column (as described above, see Example 1) HiTrap® Protein A High Performance (Merck GE17-0403-03) followed by QC by A280 / SDS-PAGE / HPLC-SEC / EU test or analysis and low endotoxin levels. Purified anti-Gliomedin was concentrated in PBS at a concentration of 1-2 mg / mL, later partially reduced with DTT at a final concentration of 10 mM. The solution was incubated at 37°C for 30 minutes followed by incubation with iodoacetamide to the at a final concentration of 20 mM for 30 minutes in the dark. The activated anti-Gliomedin was purified from excess reagents by dialysis. In parallel, a solution of MMAE in buffer at a concentration of 1-2 mM solution was prepared which was incubated to the linker solution (maleimide- activated VC-PAB) and incubated at room temperature for 30 minutes. The MMAE-linker complex was conjugated to the antibody by incubation of the activated antibody with MMAE- linker mixture at room temperature for 2-4 hours. The antibody-drug conjugate (ADC) was purified by dialysis, for the removal of excess reagents. The ADC was later analyzed for its purity, drug loading and monomeric state.

[0285] Example 7: Evaluation of targeted delivery of an antibody-MMAE drug molecule conjugate (ADC) in vitro

[0286] An antibody drug conjugate (ADC) comprising anti-Gliomedin antibody conjugated to MMAE is contacted to the primary Schwann cells, MSC80 cells, sFN02 / 2 (ATCC, CRL-2885) or sFN96.2 neurofibromas and malignant peripheral nerve sheath tumors (MPNSTs) Schwann cell lines. As a negative control, the primary cells or / and MSC80 cells are contacted with the isotype control mouse IgG conjugated to MMAE. Seed cancer cells and healthy cells (if used) are placed in a 96-well plate at a density of 5,000-10,000 cells per well.

[0287] The cells are incubated in a humidified incubator at 37°C with 5% CO2 for 24 hours and further incubated with the ADC serial-diluted concentrations for 48 hours at the same conditions. Cell death is evaluated using the MTT assay using standard manufactures protocols and is measured by absorbance of each well at 570 nm using a microplate reader.Example 8: Evaluation of the ADC functionality in a tumor-bearing mouse xenograft model

[0288] In this experiment, sNF02.2 cells are cultured in DMEM / F12 medium supplemented with 10% FBS, 1% NEAA, 1% Glutamax, and 1% sodium pyruvate. The cells are harvested in the exponential growth phase and washed once with PBS. The cells were resuspended in amixture of Matrigel and PBS at a ratio of 1 : 1. Female athymic nude mice (6-8 weeks old) were used for the study. Anesthetized mice (using a ketamine / xylazine mixture) were performed with a small incision in the left flank and their sciatic nerve was exposed by carefully dissecting the surrounding muscle tissue. 25 pL of the cell suspension were loaded into a 16G blunt-tipped needle and the cell suspension was injected slowly into the sciatic nerve. The injection site was palpated weekly to monitor tumor growth. Tumor size and tumor growth inhibition (TGI) were measured using a caliper. All tumors were resected after the treatment for 6 weeks.ReferencesLehmann and Hoke, CNS Neurol Disord Drug Targets. 2010 Dec, 9(6): 801-806.Kamil K, et al. Front. Neurol. 2019,10:87.Kresak, J.L, and Walsh, M., J Pediatr Genet. 2016, 5: 98.Chiara Pisciotta, Paola Saveri & Davide Pareyson (2021) Updated review of therapeutic strategies for Char cot-Marie-Tooth disease and related neuropathies, Expert Review of Neurotherapeutics, 21:6, 701-713, DOI: 10.1080 / 14737175.2021.1935242.Pisciotta, C.; Saveri, P.; Pareyson, D. Challenges in Treating Charcot-Marie-Tooth Disease and Related Neuropathies: Current Management and Future Perspectives. Brain Sci. 2021, 11, 1447. doi. org / 10.3390 / brainsci 11111447.Matsuda, Y., Mendelsohn, BA., Chem Pharm Bull (Tokyo). 2021 ;69, 976.Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, NY).Current Protocols in Molecular Biology, Ausubel et al., Eds (1994) Current Protocols, (Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.).Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, (Elsevier, New York).Eshed et al., Neuron, volume 47, issue 2, p215-229.Mancarella, C., et al., Int J Mol Sci., 2023, 24, 16346.Sandy et al., Mammalian RNAi: a practical guide, BioTechniques, VOL. 39, NO. 2 REVIEW.Kalia., J et.al. Curr. Org. Chem., 2010 Jan; 14(2): 138-147 doi: 10.2174 / 138527210790069839.Harmer and Samuel (1989) J. Immunol. Meth. 122(1): 115-221Perez et al. (2013) Drug Discov. Today, 1-13.Doronina et al. 92013) Nat. Biotechnol. 21(7): 778-784.Saito et al. (2013) Adv. Drug Deliv. Rev. 55(2): 199-215.Burke et al. (2009) Bioconjug. Chem. 20(6): 1242-1250.Proft T. Sortase-mediated protein ligation: an emerging biotechnology tool for protein modification and immobilization. Biotechnol Lett. 2010, 32(1): 1-10.Dubowchik et al. (2002) Bioconjug. Chem. 13(4): 855-869.Dubowchik et al. (2002) Bioorg. Med. Chem. Lett. 12(11): 1529-1532.Doronina et al. (2003) Nat. Biotechnol. 21(7): 778-784.Jeffrey et al. (2006) Bioconjug. Chem. 17(3): 831-840.Chen at al., (2013) Adv Drug Deliv Rev. 65(10): 1357-1369.Verkade, J. M. M. et al., “A Polar Sulfamide Spacer Significantly Enhances the Manufacturability, Stability, and Therapeutic Index of Antibody -Drug Conjugates”, Antibodies, 2018, 7, 12.Debinski et al. (1993) J. Biol. Chem., 268: 14065-14070.Kreitman and Pastan (1993) Bioconjug. Chem., 4: 581-585.Buchner, et al. (1992) Anal. Biochem., 205: 263-270.Narang et al. (1979) Meth. Enzymol. 68: 90-99.Brown et al. (1979) Meth. Enzymol. 68: 109-151.Beaucage et al. (1981) Tetra. Lett., 22: 1859-1862.R. Scopes (1982) Protein Purification, Springer- Ver lag, N.Y.Deutscher (1990) Methods in Enzymology Vol. 182: Guide to Protein Purification., Academic Press, Inc. N.Y.Matsuda, Y., Mendelsohn, BA., Chem Pharm Bull (Tokyo). 2021 ;69, 976.Remington: The Science and Practice of Pharmacy, 21st Ed., Gennaro, Ed., Lippencott Williams & Wilkins (2005).The United States Pharmacopeia: The National Formulary (USP 36 NF31), published in 2013.Mancarella, C., et al., Int J Mol Sci., 2023, 24, 16346.Pitiot et al. (2022) Antibodies (Basel) 11(3): 56.Brown, R., Curr Oncol Rep., 2023,12, 1409.Tamura, R. et al., Int J Mol Sci., 2022, 23, 5462.Molecular Cloning: A Laboratory Manual, J. Sambrook, etal., eds., Fourth Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 2012.Current Protocols in Molecular Biology, F. M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York.Kabat E A et al., (1991).Holhger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448.Poljak, R. J., et al. (1994) Structure 2: 1121-1123.Kipriyanov, S. M., et al. (1995) Human Antibodies and Hybridomas 6:93-101.Kipriyanov, S. M., et al. (1994) Mol. Immunol. 31: 1047-1058.

Claims

CLAIMS1. A drug conjugate comprising a targeting moiety conjugated to a drug molecule, wherein the targeting moiety binds to a cell adhesion moiety and / or receptor expressed on a Schwann cell to mediate targeted delivery of the drug conjugate to the Schwann cell, and wherein the drug molecule modifies expression or activity of a disease-associated molecule, and / or confers a cytotoxic effect, in the Schwann cell.

2. The drug conjugate of claim 1, wherein the cell adhesion moiety and / or receptor is selected from a group consisting of Gliomedin, a leprosy receptor e.g., Laminin alpha 2-G4-5 (LNa2G), alpha- Dystroglycan (alpha-DG) and Myelin Protein zero (PO protein), TAM (Tyro3, Axl, Mer) receptor, cell adhesion molecule (Cadm), or any Schwann cell specific receptor.

3. The drug conjugate of claim 1, wherein the cell adhesion moiety and / or receptor is selected from Nectin-like protein (Neel) e.g. Necll, Necl3, Necl2 and Necl4, SynCAM e.g. SynCAM4, Cadm e.g. Cadml-Cadm3, Neurofascinl55 (NF155), TAG1, myelin-associated glycoprotein (MAG), and neuronal cell adhesion molecule (NrCAM).

4. The drug conjugate of claim 1, wherein the targeting moiety is an antibody or antigen binding fragment thereof or a ligand molecule.

5. The drug conjugate of claim 1, wherein the targeting moiety is a ligand molecule, and wherein the ligand molecule comprises a protein, peptide, a glycoprotein, a glycan, a carbohydrate moiety, a fatty acid, a dendrimer, or a synthetic small molecule.

6. The drug conjugate of any one of claims 1-5, wherein the targeting moiety is selected from antihuman Gliomedin Antibody, anti-Cadm4 mAb244 / 5 (NeuroMAB), Necl4-Fc antibody, anti- SynCAM4 Antibody, IGSF4C / SynCAM4 Antibody, neurofascin-186 (NF186) or a portion or derivative thereof, glycolipid PGL-1 or a portion or derivative thereof, trisaccharide of PGL-1 or a portion or derivative thereof, ML-LBP21 (histone-like protein / Hlp) or a portion or derivative thereof.

7. The drug conjugate of claim 4, wherein the antibody or antigen binding fragment thereof comprises IgG (Immunoglobulin G), including IgGl, IgG2a IgG2b, IgG3 and IgG4, IgA (Immunoglobulin A), including IgAl and IgA2, IgM (Immunoglobulin M), IgE (Immunoglobulin E), IgD (Immunoglobulin D), fragment antigen-binding (Fab fragment), fragment crystallizable region (Fc region), monovalent Fab’, divalent Fab2, single-chain variable fragment (scFv), diabody, minibody, nanobody, single-domain antibody (sdAb), (scFv)2, Fab,Fab', F(ab')2, Fv, dAb, Fd fragments, diabodies, F(ab')3, disulfide linked Fv, sdAb (VHH or nanobody), CDR (Complementarity-determining region), di-scFv, bi-scFv, tascFv (tandem scFv), triabody, tetrabody, V-NAR domain, Fcab, IgGACH2, DVD-Ig, probody, a DARPin, a Centyrin, an affibody, an affilin, an affitin, an anticalin, an avimer, a Fynomer, a Kunitz domain peptide, a monoclonal antibody, a monobody (or adnectin), a tribody, and a nanofitin and miniproteins or camelid antibody, or a monospecific, bispecific, trispecific, or a multi-specific antibody or binding fragment or antibody-mimetic thereof.

8. The drug conjugate of claim 4, wherein the antibody or antigen binding fragment thereof comprises binding specificity to Gliomedin protein.

9. The drug conjugate of claim 8, wherein the antibody or antigen binding fragment thereof comprises a complementary determining region (CDR) sequence having at least 90%, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity to a sequence selected from SEQ ID NO: 3-22 and 30 or a fragment, functional variant, or a combination thereof.

10. The drug conjugate of claim 8, wherein the antibody or antigen binding fragment thereof comprises a heavy chain variable region (VH) comprising heavy chain complementarity determining region (CDR) selected from CDR-H1, CDR-H2 and CDR-H3 or any combination thereof, and / or a light chain variable region (VL) comprising light chain CDR selected from CDR-L1, CDR-L2, and CDR-L3, or any combination thereof, and wherein the CDR-H1, CDR- H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, respectively, comprise a sequence having at least 90% identity to a sequence selected from:(a) the amino acid sequences of CDR-H1 comprising SEQ ID NO: 3, 4, 9 and 10;(b) the amino acid sequences of CDR-H2 comprising SEQ ID NO: 5, 6, 11 and 12;(c) the amino acid sequences of CDR-H3 comprising SEQ ID NO: 7, 8, 13 and 14;(d) the amino acid sequences of CDR-L1 comprising SEQ ID NO: 15, 16 and 20;(e) the amino acid sequences of CDR-L2 comprising SEQ ID NO: 17, 18, 21 and 30;(f) the amino acid sequences of CDR-L3 comprising SEQ ID NO: 19 and 22; or any functional variant, fragment or a combination thereof.

11. The drug conjugate of any one of claims 1-10, wherein the drug molecule comprises an oligonucleotide, a peptide, a protein, an immunoglobulin, a small molecule, or a complex or any combination thereof.

12. The drug conjugate of any one of claims 1-11, wherein the drug molecule comprises a degrader molecule (such as a protein degrader), a toxin such as Auristatin derivative, Maytansinoids, Calicheamicins, Pyrrolobenzodiazepines, Duocarmycins, Topoisomerase Inhibitors, a- Amanitin, Epothilones, cell death inducing molecules, cytotoxic agent, and / or any combination thereof.

13. The drug conjugate of any one of claims 1-11, wherein the drug molecule comprises enzymes, enzyme inhibitors, proteins, kinase and / or phosphatase inhibitors or any protein-specific modulating agent.

14. The drug conjugate of any one of claims 1-11, wherein the drug molecule comprises a doublestranded RNAi molecule or a single-stranded antisense oligonucleotide.

15. The drug conjugate of any one of claims 1-11, wherein the drug molecule comprises RNA molecule such as tRNA, rRNA and mRNA, coding (cRNA), noncoding RNA (ncRNA), micro RNA (miRNA), small nucleolar RNA (snoRNA), small nuclear RNA (snRNA), smallinterfering RNA (siRNA), PlWI-interacting RNA (piRNA), short hairpin RNA (shRNA), microRNA (miRs), a double-stranded RNAi molecule, a single-stranded antisense oligonucleotide, antisense oligonucleotides (ASOs) and antiparallel triplex forming oligonucleotides.

16. The drug conjugate of claim 15, wherein the drug molecule comprises siRNA molecule comprising a nucleic acid sequence which has at least 95%, such as at least 96%, 97%, 98% or at least 99% sequence identity with a sequence selected from SEQ ID NO: 25-29, a complementary sequence thereof, or any combination thereof.

17. The drug conjugate of any one of claims 1-16, wherein the drug molecule is conjugated with the targeting moiety via a linker.

18. The drug conjugate of claim 17, wherein the linker is a cleavable linker, a non-cleavable linker, covalently bound linker such as by chemical conjugation, linkers based on non-covalent interactions, a carbohydrate linker, a peptide linker, a glycan linker, or a non-peptide linker. Examples for such linkers include, but not limited to, Hydrazone Linkers, Disulfide Linkers, Peptide Linkers (Valine-citrulline (Val-Cit) and Alanine-alanine (Ala-Ala)), Beta-Glucuronide Linkers, Thioether Linkers (such as SMCC (succinimidyl-4-(N-maleimidomethyl)cyclohexane- 1 -carboxylate)), PEG Linkers, Conditional Linkers (such as Azobenzene Linkers and Diselenide Linkers).

19. The drug conjugate of any one of claims 1-18, wherein the Drug molecule to the Targeting Moiety Ratio (DTMR) is in the range of 1 to 10 or characterized by a DTMR between 2 to 8, such as DTMR between 2 to 5.

20. The drug conjugate of any one of claims 1-19, wherein the disease is selected from peripheral nervous system (PNS) disorders related to dysfunction and / or degeneration of Schwann cells and / or axons, and axon related disorders.

21. The drug conjugate of any one of claims 1-11, wherein the disease is selected from peripheral demyelinating disease, peripheral neuropathy and / or hereditary motor and sensory neuropathy such as Charcot-Marie-Tooth disease (CMT), Guillain-Barre syndrome (acute inflammatory demyelinating polyradiculopathy type), chronic inflammatory demyelinating polyneuropathy, nerve trauma, neuropathic pain, post-chemotherapy neuropathy, diabetic neuropathy, diabetic peripheral neuropathy, migraine, fibromyalgia, demyelinating diseases such as multiple sclerosis, and malignant peripheral nerve sheath tumors (MPNSTs), endometriosis abdominopelvic pain and nerve injury.

22. The drug conjugate of claim 21, wherein the disease is Charcot-Marie-Tooth disease type 1 (CMT1), such as CMT type 1A (CMT1 A).

23. The drug conjugate of claim 12, wherein the disease is a nerve tumor disease such as schwannomatosis, schwannomas and neurofibromatosis type 1 (NF1).

24. The drug conjugate of any one of claims 1 -23, wherein the disease-associated molecule is at least one of peripheral myelin protein 22 (PMP22), SMARCB 1 encoded SWI / SNF subunit, LZTR1 , chronic inflammatory demyelinating polyneuropathy (CIDP), Histone deacetylase-6 (HDAC6), P2X7 receptors, Neurotrophin-3 (NT-3), antiganglioside antibodies, enzymes, phosphatases, kinases, receptor tyrosine kinases (RTKs), Nonreceptor protein tyrosine kinases (nrPTKs), PMP2, MPZ, LITAF, FBLN5, EGR2, HDAC6, NEFL, GDAP1, MTMR2, SH3TC2, NDRG1, PRX, FGD4, SBF1, SBF2, FIG4, CTDP1, SURF1, ADCY6, CNTNAP1, HK1, MFN2, LRSAM1, NEFH, KIF5A, ATP1A1, VCP, TFG, DHTKD1, TUBB3, NAGLU, DCAF8, DGAT2, M0RC2, HSPB1, HSPB3, HSPB8, GARS, AARS, HARS, MARS, DYNC1H1, BICD2, REEP1, BSCL2, SETX, SLC5A7, MYH14, TRPV4, RAB7LMNA, PNKP, TRIM2, SPG11, MME, MCM3AP, SLC25A46, SCO2, MPV17, C12 or f65, IGHMBP2, SIGMAR1, VRK1, ATP7A, UBA1, GLE1, LAS1L, GJB1, YARS, INF2, DRP2, DNM2, GNB4, PDK3, GDAP1, COX6A1, PLEKHG5, KARS, AIFM1 and PRPS1.

25. A pharmaceutical composition comprising a drug conjugate of any one of claims 1-24 and a pharmaceutically acceptable excipient.

26. The pharmaceutical composition of claim 25, wherein the pharmaceutical composition is formulated for parenteral, systemic, intravenous, subcutaneous, or intrasciatic delivery.

27. A method of targeted delivery of a drug to a Schwann cell, comprising allowing contacting of the drug conjugate of any one of claims 1-24 or the pharmaceutical composition of any one of claims 25-26 to the Schwann cell, wherein the drug molecule comprises the drug.

28. The method of claim 27, wherein the drug is internalized into the Schwann cell upon binding of the targeting moiety to the cell adhesion moiety and / or receptor on the Schwann cell.

29. A method of treating or diagnosing a subject with a disease associated with dysfunction and / or degeneration of Schwann cells and / or axons, and axon related disorders, comprising administering to a subject in need of such a treatment or diagnosis an effective amount of the drug conjugate according to any one of claims 1-24 or a pharmaceutical composition according to claim 25 and 26.

30. The method of claim 29, wherein the disease is selected from schwannomatosis, schwannomas and neurofibromatosis type 1 (NF1) and the drug molecule is a degrader molecule (such as a protein degrader), a toxin such as Auristatin derivative, Maytansinoids, Calicheamicins, Pyrrolobenzodiazepines, Duocarmycins, Topoisomerase Inhibitors, a-Amanitin, Epothilones, cell death inducing molecules, cytotoxic agent, and / or any combination thereof.

31. The method of claim 29, wherein the disease is selected from peripheral demyelinating disease, peripheral neuropathy and / or hereditary motor and sensory neuropathy such as Charcot-Marie- Tooth disease (CMT), Guillain-Barre syndrome (acute inflammatory demyelinating polyradiculopathy type), chronic inflammatory demyelinating polyneuropathy, nerve trauma, neuropathic pain, post-chemotherapy neuropathy, diabetic neuropathy, diabetic peripheral neuropathy, migraine, fibromyalgia, demyelinating diseases such as multiple sclerosis, and malignant peripheral nerve sheath tumors (MPNSTs), endometriosis abdominopelvic pain and nerve injury, and the drug molecule is an oligonucleotide, a peptide, a protein, an immunoglobulin.

32. The method of claim 31, wherein the drug molecule comprises siRNA molecule comprising a nucleic acid sequence which has at least 95%, such as at least 96%, 97%, 98% or at least 99% sequence identity with a sequence selected from SEQ ID NO: 25-29, a complementary sequence thereof, or any combination thereof.

33. An antibody or antigen binding fragment thereof having an anti-Gliomedin activity comprising a complementary determining region (CDR) sequence having at least 90%, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity to a sequence selected from SEQ ID NO: 3-22 and 30 or a fragment, functional variant or a combination thereof.

34. The antibody or antigen binding fragment thereof of claim 33, wherein the antibody or antigen binding fragment thereof comprises binding specificity to Gliomedin protein.

35. The antibody or antigen binding fragment thereof of any one of claims 33-34, wherein the antibody or antigen binding fragment thereof comprises a heavy chain variable region (VH) comprising heavy chain complementarity determining region (CDR) selected from CDR-H1, CDR-H2 and CDR-H3 or any combination thereof, and / or a light chain variable region (VL) comprising light chain CDR selected from CDR-L1, CDR-L2, and CDR-L3, or any combination thereof, and wherein the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, respectively, comprise a sequence having at least 90%, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to a sequence selected from:(a) the amino acid sequences of CDR-H1 comprising SEQ ID NO: 3, 4, 9 and 10;(b) the amino acid sequences of CDR-H2 comprising SEQ ID NO: 5, 6, 11 and 12;(c) the amino acid sequences of CDR-H3 comprising SEQ ID NO: 7, 8, 13 and 14;(d) the amino acid sequences of CDR-L1 comprising SEQ ID NO: 15, 16 and 20;(e) the amino acid sequences of CDR-L2 comprising SEQ ID NO: 17, 18, 21 and 30;(f) the amino acid sequences of CDR-L3 comprising SEQ ID NO: 19 and 22; or any fragment or a combination thereof.

36. The antibody or antigen binding fragment thereof of any one of claim 33-35, wherein the antibody or antigen binding fragment thereof comprises an amino acid sequence which has at least 90%, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity with SEQ ID NO:1, SEQ ID NO:2 or a combination thereof.

37. Use of an amino acid sequence comprising a complementary determining region (CDR) sequence having at least 90%, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% sequence identity to a sequence selected from SEQ ID NO: 3-22 and 30 or a fragment, functional variant or a combination thereof, as an anti-Gliomedin antibody or antigen binding fragment thereof.

38. The use of claim 37, wherein the antibody or antigen binding fragment thereof comprises binding specificity to Gliomedin protein.

39. The use of any one of claims 37-38, wherein the antibody or antigen binding fragment thereof comprises a heavy chain variable region (VH) comprising heavy chain complementarity determining region (CDR) selected from CDR-H1, CDR-H2 and CDR-H3 or any combination thereof, and / or a light chain variable region (VL) comprising light chain CDR selected from CDR-L1, CDR-L2, and CDR-L3, or any combination thereof, and wherein the CDR-H1, CDR- H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, respectively, comprise a sequence having at least 90%, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to a sequence selected from:(a) the amino acid sequences of CDR-H1 comprising SEQ ID NO: 3, 4, 9 and 10;(b) the amino acid sequences of CDR-H2 comprising SEQ ID NO: 5, 6, 11 and 12;(c) the amino acid sequences of CDR-H3 comprising SEQ ID NO: 7, 8, 13 and 14;(d) the amino acid sequences of CDR-L1 comprising SEQ ID NO: 15, 16 and 20;(e) the amino acid sequences of CDR-L2 comprising SEQ ID NO: 17, 18, 21 and 30;(f) the amino acid sequences of CDR-L3 comprising SEQ ID NO: 19 and 22; or any fragment or a combination thereof.

40. The use of any one of claims 37-39, wherein the antibody or antigen binding fragment thereof comprises at least 90%, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity with SEQ ID NO:1, SEQ ID NO:2 or a combination thereof, as an anti-Gliomedin antibody or antigen binding fragment thereof.

41. A nucleic acid encoding the antibody or antigen binding fragment thereof of any one of claims 33-36.

42. A host cell comprising the nucleic acid of claim 41.

43. A method for producing the antibody or antigen binding fragment thereof of any one of claims 33-36, which comprises the steps of culturing the host cell of claim 42, and recovering the polypeptides from the cell culture.

44. An isolated polynucleotide having siRNA activity for reducing human PMP22 expression, comprising a nucleic acid sequence which has at least 95%, such as at least 96%, 97%, 98% or at least 99% identity with SEQ ID NO:25-29 or a combination thereof.

45. Use of a nucleic acid sequence comprising at least 95%, such as at least 96%, 97%, 98% or at least 99% identity with SEQ ID NO:25-29 or a combination thereof, as siRNA molecule for reducing PMP22 expression in a Schwann cell.

46. A composition comprising the antibody or antigen binding fragment thereof of any one of claims 33-36 and / or the isolated polynucleotide of claim 44 and a pharmaceutically acceptable carrier.

47. A drug conjugate comprising a targeting moiety conjugated to a drug molecule, wherein the targeting moiety binds to a cell adhesion moiety and / or receptor expressed on a Schwann cell to mediate targeted delivery of the drug conjugate to the Schwann cell, and the drug molecule modifies expression or activity of a disease-associated molecule, and / or confers a cytotoxic effect, in the Schwann cell, wherein the targeting moiety comprises the antibody or antigenbinding fragment of any one of claims 33-36.

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