Targeted delivery of schwann cells and methods of treatment of schwann cell related diseases

Abstract

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

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 388,543, filed on July 12, 2022, the entire contents of which are incorporated herein by reference. Background Art

[0003] Schwann cells, also called neurolemmal cells, produce myelin sheaths around neuronal axons in the peripheral nervous system. They play a key role in the pathology of various inflammatory, metabolic or hereditary neuropathies (see, e.g., Lehmann and CNS Neurol Disord Drug Targets. 2010 Dec, 9(6): 801–806; Kamil K, et al. Front. Neurol. 2019, 10: 87, the entire contents of which are incorporated herein by reference).

[0004] Some peripheral neuropathies are associated with abnormal protein or gene expression in Schwann cells, and inhibition or regulation of such protein or gene expression in Schwann cells has been considered to treat those peripheral neuropathies. However, currently practiced drug delivery methods, such as delivery via nanoparticles and AAV, have disadvantages, including non-specific targeting, reduced targeting efficiency, or safety issues. Therefore, there is a need to specifically target Schwann cells to manage, treat, or alleviate a large repertoire of Schwann cell-related diseases. Summary of the invention

[0005] Recognizing the need for more effective and targeted delivery of therapeutic compositions to Schwann cells, in one aspect, the present disclosure provides a drug conjugate comprising a targeting moiety conjugated to a drug molecule, wherein the targeting moiety binds to a receptor expressed on Schwann cells to mediate targeted delivery of the drug conjugate to Schwann cells, and wherein the drug molecule modifies the expression or activity of a disease-associated molecule in the Schwann cells.

[0006] In some instances, the receptor is selected from the group consisting of: a leprosy receptor (e.g., laminin 2, P0 protein), a neuromodulatory peptide (gliomedin), or a cell adhesion molecule (Cadm). In some instances, the receptor is a neuromodulatory peptide. In some instances, the receptor is a Cadm. In some instances, the receptor is a leprosy receptor.

[0007] In some instances, the targeting moiety is an antibody or its antigen binding fragment or a ligand molecule. In some instances, the antibody or its antigen binding fragment comprises a monovalent Fab', a divalent Fab2, a single chain variable fragment (scFv), a double antibody, a mini antibody, a nano antibody, a single domain antibody (sdAb) or a camel antibody or its binding fragment or an antibody mimetic. In some instances, the targeting moiety is a ligand molecule, and wherein the ligand molecule comprises a peptide, a glycoprotein, a carbohydrate moiety, a dendrimer or a synthetic small molecule.

[0008] In some instances, the drug molecule comprises a peptide or a small molecule. In some instances, the drug molecule comprises a double-stranded RNAi molecule or a single-stranded antisense oligonucleotide.

[0009] In some instances, the drug molecule is conjugated to the targeting moiety via a linker. In some instances, the linker is a cleavable linker or a non-cleavable linker. In some instances, the ratio of the drug molecule to the targeting moiety is 1:1, 2:1 or 1:2.

[0010] In some aspects, provided herein is a pharmaceutical composition comprising a drug conjugate as described herein and a pharmaceutically acceptable excipient. In some examples, the pharmaceutical composition is formulated for parenteral, intravenous, subcutaneous, intrathecal or intrasciatic injection.

[0011] In some aspects, the present invention provides a method for targeted delivery of a drug to Schwann cells, comprising contacting a drug conjugate described herein or a pharmaceutical composition described herein with a Schwann cell, wherein the drug molecule comprises a drug or a precursor of a drug. In some examples, when the targeting moiety binds to a receptor on a Schwann cell, the drug or the precursor of the drug is internalized into the Schwann cell and exerts the biological activity of the drug or the precursor of the drug.

[0012] Imported by reference

[0013] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated herein by reference. To the extent that publications and patents or patent applications incorporated by reference conflict with the disclosure contained in this specification, this specification is intended to supersede and / or take precedence over any such conflicting material. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] This application contains at least one drawing in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0015] The novel features of the present disclosure are specifically described in the appended claims. The features and advantages of the present disclosure will be better understood by referring to the following detailed description and the accompanying drawings (also referred to as Figures and FIG.), which describe illustrative embodiments utilizing the principles of the present disclosure, and in the accompanying drawings:

[0016] Figure 1A-1F Several targeting moieties, including an anti-neuromedin antibody (mAb94), were demonstrated to be internalized into Schwann cells in vitro. Figure 1A Illustrated are rat Schwann cell cultures incubated with pre-labeled (red) anti-nectin-like protein 4 (Necl4, also known as Cadm4) antibody (mAb244 / 5). Figure 1B Illustrated are cultures of rat Schwann cells incubated with pre-labeled (red) mouse anti-neuromedulin antibody (mAb94). Figure 1C Illustrated are cultures of rat Schwann cells incubated with pre-labeled (red) mouse anti-myelin associated glycoprotein (MAG) antibody (mAb513). Figure 1D Internalization of an anti-neuromedulin antibody (mAb94) in rat Schwann cell cultures at different incubation times is illustrated. Figure 1E Co-localization of internalized anti-neuromedulin antibody (mAb94) and lysosomal markers in rat Schwann cell cultures is demonstrated. Figure 1F Internalization of an anti-neuromedin antibody (mAb94) covalently conjugated to Cyanine 5 NHS-ester in rat Schwann cell culture is demonstrated.

[0017] Figure 2A-2C It was demonstrated that an anti-neuromedin antibody (mAb94) was specifically internalized into Schwann cells in mixed culture and recognized both rat and human neuromedin. Figure 2A It was demonstrated that an anti-neuromedulin antibody (mAb94) was specifically internalized into wild-type Schwann cells in a mixed culture with neurons and Schwann cells, but not into mutant Schwann cells without neuromedin expression. Figure 2B It was demonstrated that an anti-neuromedulin antibody (mAb94) was specifically internalized into wild-type Schwann cells in a mixed culture with neurons and Schwann cells, but not into mutant Schwann cells without neuromedin expression. Figure 2C It was demonstrated that in a mixed culture of neurons, Schwann cells and fibroblasts, an anti-neuromedulin antibody (mAb94) specifically bound to wild-type Schwann cells but not to mutant Schwann cells that did not express neuromedin.

[0018] Figure 3 Internalization of an anti-neuromedin antibody (mAb94) into COS7 cells transfected with rat or human neuromedin is demonstrated.

[0019] Figures 4A-4E The internalization of an anti-neuromedulin antibody (mAb94) into Schwann cells in vivo and its distribution in different organs are demonstrated. Figure 4A Illustrated are sciatic nerves harvested from mice #1-3 injected with 650-mAb94 antibody and mice #7-9 injected with control 650-IgG. Both groups were sacrificed 24 hours after injection. Figure 4B Illustrated are sciatic nerves harvested from mice #4-6 injected with 650-mAb94 antibody and mice #10-12 injected with control 650-IgG. Both groups were sacrificed 48 hours after injection. Figure 4C Depicted is a comparison between the sciatic nerves of mice injected with control 650-IgG (#12) and uninjected mice (#13). Figure 4D Depicted are fluorescence images of Cy5-labeled anti-neuromedulin antibody (mAb94) (far red) in different organs 24 hours after intravenous injection. Figure 4E Depicted are fluorescence images of Cy5-labeled anti-neuromedulin antibody (mAb94) (far red) in different organs 48 hours after intravenous injection. DETAILED DESCRIPTION

[0020] Provided herein are drug conjugates comprising a targeting moiety conjugated to a drug molecule, wherein the targeting moiety binds to a receptor expressed on a Schwann cell to mediate targeted delivery of the drug conjugate to the Schwann cell, and wherein the drug molecule modifies the expression or activity of a disease-associated molecule in the Schwann cell. Also disclosed herein are methods for targeted delivery of a drug to Schwann cells. Also disclosed herein are methods for delivering a drug to Schwann cells by conjugating the drug to a targeting moiety that specifically or preferentially targets Schwann cells. Also disclosed herein are methods for treating or preventing peripheral demyelinating disease or peripheral neuropathy in a subject in need thereof, or for alleviating or reducing the symptoms of a peripheral demyelinating disease or peripheral neuropathy.

[0021] Conjugate

[0022] Targeting moiety

[0023] Historically, treatment of peripheral nervous system (PNS) disorders associated with, for example, axonal loss, neuronal axonal dysfunction, or abnormal axon-Schwann cell interactions has focused on targeting axons, but treatment has been shown to have poor clinical outcomes. For certain PNS disorders in which axonal dysfunction is associated with Schwann cell loss, or directly or indirectly with Schwann cell dysfunction, targeting drugs specifically or preferentially to Schwann cells may increase the efficacy and outcome of treatment.

[0024] However, the specificity of the manipulation and / or modification of Schwann cells is a major obstacle to the advancement of Schwann cell therapy. To meet this need, in some examples herein, a targeting moiety is provided that specifically directs cargo to Schwann cells.

[0025] In some aspects, the targeting moiety disclosed herein is combined with a receptor expressed on Schwann cells. In some aspects, the targeting moiety disclosed herein is combined with a part of a receptor complex expressed on Schwann cells. In some instances, the receptor is a transmembrane molecule comprising an extracellular domain, and the targeting moiety is combined with the extracellular domain of the receptor. In some instances, the receptor complex comprises an extracellular molecule that directly or indirectly forms a complex with a receptor molecule (e.g., a membrane-bound receptor via a transmembrane domain). In some instances, the targeting moiety is combined with the extracellular molecule of the complex. In some instances, the targeting moiety is combined with a cell adhesion molecule that mediates the interaction between myelinated Schwann cells and axons in their sheaths. In some instances, the receptor is expressed in myelinated Schwann cells. In some instances, the receptor is highly expressed at the edge of myelinating cells. In some instances, the receptor mediates the interaction between axons and glial cells, or mediates axon-glial contact. In some instances, the receptor mediates the uptake of Schwann cells by pathogens (or parts thereof) in certain disease states. Thus, as used herein, a targeting moiety that binds to a receptor also includes a targeting moiety that binds to a portion of a receptor complex that naturally occurs in Schwann cells in healthy or pathological conditions.

[0026] In some instances, the targeting moiety disclosed herein binds to a leprosy receptor. In a specific instance, the targeting moiety disclosed herein binds to laminin 2. In a specific instance, 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 α2-subunit of laminin 2. In some instances, the targeting moiety disclosed herein binds to the G domain of the α2-subunit of laminin 2. In some instances, the targeting moiety disclosed herein binds to the G1 domain of the α2-subunit of laminin 2. In some instances, the targeting moiety disclosed herein binds to the G2 domain of the α2-subunit of laminin 2. In some instances, the targeting moiety disclosed herein binds to the G3 domain of the α2-subunit of laminin 2. In some instances, the targeting moiety disclosed herein binds to the G4 domain of the α2-subunit of laminin 2. In some instances, the targeting moiety disclosed herein binds to the G5 domain of the α2-subunit of laminin 2. In some instances, the targeting moiety disclosed herein binds to the β1-subunit of laminin 2. In some instances, the targeting moiety disclosed herein binds to the γ1-subunit of laminin 2. In some instances, the targeting moiety disclosed herein binds to the amino acids 2901-3106 of lamin-2. In other instances, the targeting moiety disclosed herein binds to the P0 protein (myelin protein zero). In some instances, the targeting moiety disclosed herein binds to the N-terminal extracellular immunoglobulin (Ig)-like domain of the 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 of its subunits, and / or adds sulfate, acyl and phosphate groups.

[0027] In some instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof that binds to a leprosy receptor. In a specific instance, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof that binds to laminin 2. In a specific instance, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof that 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 that binds to the α2-subunit of laminin 2. In some instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof that binds to the G domain of the α2-subunit of laminin 2. In some instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof that binds to the G1 domain of the α2-subunit of laminin 2. In some instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof that binds to the G2 domain of the α2-subunit of laminin 2. In some examples, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof that binds to the G3 domain of the α2-subunit of laminin 2. In some examples, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof that binds to the G4 domain of the α2-subunit of laminin 2. In some examples, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof that binds to the G5 domain of the α2-subunit of laminin 2. In some examples, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof that binds to the β1-subunit of laminin 2. In some examples, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof that binds to the γ1-subunit of laminin 2. In some examples, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof that binds to amino acids 2901-3106 of lamin-2. In other examples, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof that binds to P0 protein (myelin protein zero). In some instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof that binds to the N-terminal extracellular immunoglobulin (Ig)-like domain of the P0 protein. In some instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof that binds to an N-linked oligosaccharide or any subunit thereof in the extracellular domain of the leprosy receptor, and / or adds sulfate, acyl, and phosphate groups.

[0028] 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 the anti-laminin antibody ABIN7439129. In some instances, the targeting moiety disclosed herein is the anti-laminin alpha-2 monoclonal antibody (Thermo Fisher, #CL3450).

[0029] In some instances, the targeting moiety disclosed herein is combined with a neuromodulatory peptide. In some instances, the targeting moiety disclosed herein is combined with the extracellular region of a neuromodulatory peptide. In some instances, the targeting moiety disclosed herein is combined with the C-terminal region of a neuromodulatory peptide. In some instances, the targeting moiety disclosed herein is combined with a portion of the C-terminal region of a neuromodulatory peptide. In some instances, the targeting moiety disclosed herein is combined with the coiled-coil domain of a neuromodulatory peptide. In some instances, the targeting moiety disclosed herein is combined with the interrupted collagen repeat domain of a neuromodulatory peptide. In some instances, the targeting moiety disclosed herein is combined with the olfactomedin domain of a neuromodulatory peptide. In some instances, the targeting moiety disclosed herein is combined with the amino acid residues 273-287 of rat neuromodulatory peptide NP_852047.2 (CVIPNDDTLVGRA). In some instances, the targeting moiety disclosed herein is combined with the amino acid residues 365-460 of a neuromodulatory peptide.

[0030] In some instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof that binds to a neuromodulatory peptide. In some instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof that binds to the extracellular region of a neuromodulatory peptide. In some instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof that binds to the C-terminal region of a neuromodulatory peptide. In some instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof that binds to a portion of the C-terminal region of a neuromodulatory peptide. In some instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof that binds to a coiled-coil domain of a neuromodulatory peptide. In some instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof that binds to an interrupted collagen repeat domain of a neuromodulatory peptide. In some instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof that binds to the olfactory mediator domain of a neuromodulatory peptide. In some instances, the targeting moiety disclosed herein is an antibody or antigen-binding fragment thereof that binds to amino acid residues 273-287 of rat neuromedin NP_852047.2 (CVIPNDDTLVGRA). In some instances, the targeting moiety disclosed herein is an antibody or antigen-binding fragment thereof that binds to amino acid residues 365-460 of neuromedin.

[0031] In some instances, the targeting moiety disclosed herein is a commercially available antibody that binds to a neuromodulin. In some instances, the targeting moiety disclosed herein is an anti-neurmodulin antibody (mAb94) disclosed in Eshed et al., Neuron, volume 47, issue 2, p215-229, the entire contents of which are incorporated herein by reference. In some instances, the targeting moiety disclosed herein is an anti-GLDN antibody NovoPro#:175453. In some instances, the targeting moiety disclosed herein is an anti-GLDN antibody Thermo Fisher#:BS-11032R.

[0032] In some instances, the targeting moieties disclosed herein bind to cell adhesion molecules (Cadm). In some instances, the targeting moieties disclosed herein bind to Cadm1. In some instances, the targeting moieties disclosed herein bind to the IgV-set domain of Cadm1, or a portion thereof. In some instances, the targeting moieties disclosed herein bind to the IgC1-set domain of Cadm1, or a portion thereof. In some instances, the targeting moieties disclosed herein bind to the IgI-set domain of Cadm1, or a portion thereof. In specific instances, the targeting moieties disclosed herein bind to Cadm2. In some instances, the targeting moieties disclosed herein bind to the IgV-set domain of Cadm2, or a portion thereof. In some instances, the targeting moieties disclosed herein bind to the IgC1-set domain of Cadm2, or a portion thereof. In some instances, the targeting moieties disclosed herein bind to the IgI-set domain of Cadm2, or a portion thereof. In some instances, the targeting moieties disclosed herein bind to Cadm3. In some instances, the targeting moieties disclosed herein bind to the IgV-set domain, or a portion thereof, of Cadm3. In some instances, the targeting moieties disclosed herein bind to the IgC1-set domain, or a portion thereof, of Cadm3. In some instances, the targeting moieties disclosed herein bind to the IgI-set domain, or a portion thereof, of Cadm3. In some instances, the targeting moieties disclosed herein bind to Cadm4. In some instances, the targeting moieties disclosed herein bind to the IgV-set domain, or a portion thereof, of Cadm4. In some instances, the targeting moieties disclosed herein bind to the IgC1-set domain, or a portion thereof, of Cadm4. In some instances, the targeting moieties disclosed herein bind to the IgI-set domain, or a portion thereof, of Cadm4.

[0033] In some instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof that binds to a cell adhesion molecule (Cadm). In some instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof that binds to Cadm1. In some instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof that binds to the IgV-set domain or a portion thereof of Cadm1. In some instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof that binds to the IgC1-set domain or a portion thereof of Cadm1. In some instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof that binds to the IgI-set domain or a portion thereof of Cadm1. In specific instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof that binds to Cadm2. In some instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof that binds to the IgV-set domain or a portion thereof of Cadm2. In some instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof that binds to the IgC1-set domain or a portion thereof of Cadm2. In some instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof that binds to the IgI-set domain or a portion thereof of Cadm2. In some instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof that binds to Cadm3. In some instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof that binds to the IgV-set domain or a portion thereof of Cadm3. In some instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof that binds to the IgC1-set domain or a portion thereof of Cadm3. In some instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof that binds to the IgI-set domain or a portion thereof of Cadm3. In some instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof that binds to the IgI-set domain or a portion thereof of Cadm3. In some instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof that binds to Cadm4. In some instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof that binds to the IgV-set domain or a portion thereof of Cadm4. In some instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof that binds to the IgC1-set domain or a portion thereof of Cadm4. In some instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof that binds to the IgI-set domain or a portion thereof of Cadm4.

[0034] In some instances, the targeting moiety disclosed herein is a commercially available antibody that binds to Cadm1. 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 Necl4–Fc antibody from Eshed et al., Neuron, volume 47, issue 2, p215-229. In some instances, the targeting moiety disclosed herein is an anti-SynCAM4 antibody (Biolegand, #833302). In some instances, the targeting moiety disclosed herein is an IGSF4C / SynCAM4 antibody (rndsystems, #MAB41642).

[0035] In some instances, the targeting moiety directs the conjugate to Schwann cells, and the conjugate is internalized after the targeting moiety binds to the receptor, which is about 24 hours, about 36 hours, about 48 hours, about 60 hours, or about 72 hours after contacting the Schwann cells. In some instances, the targeting moiety directs the conjugate to Schwann cells, and the conjugate is internalized after the targeting moiety binds to the receptor, which is about 24 hours, about 36 hours, about 48 hours, about 60 hours, or about 72 hours after contacting the Schwann cells. 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 cells. In some examples, the targeting moieties disclosed herein direct and internalize the conjugates disclosed herein in 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 cells.

[0036] In some instances, the drug molecules disclosed herein are released after internalization into Schwann cells. In some instances, the drug molecules disclosed herein are released after the conjugates disclosed herein enter the lysosomes of Schwann cells. In some instances, the drug molecules disclosed herein are released into the cytoplasm of Schwann cells. In some instances, the drug molecules disclosed herein are released into the nucleus of Schwann cells.

[0037] In some aspects, the targeting moiety disclosed herein is an antibody or an antigen-binding fragment thereof. In some instances, the antibody or its antigen-binding fragment comprises a monovalent Fab', a bivalent Fab2, a single-chain variable fragment (scFv), a diabody, a minibody, a nanobody, a single domain antibody (sdAb) or a camelid antibody or a binding fragment thereof. In other examples, the targeting moieties disclosed herein are (scFv)2, Fab, Fab', F(ab')2, Fv, dAb, Fd fragment, diabody, F(ab')3, disulfide-linked Fv, sdAb (VHH or nanobody), CDR, di-scFv, bi-scFv, tascFv (tandem scFv), triabody, tetrabody, V-NAR domain, Fcab, IgGACH2, DVD-Ig, probody, DARPin, Centyrin, affibody, affilin, affitin, anticalin, avimer, Fynomer, Kunitz domain peptide, monobody (or adnectin), tribody, and nanofitin and mini-protein. In other examples, the targeting moieties disclosed herein are variants and derivatives of antibodies, including functional fragments of antibodies that retain the ability to bind to receptors expressed on Schwann cells.Exemplary functional fragments include Fab fragments (e.g., antibody fragments containing an antigen binding domain and comprising a portion of a light chain and a heavy chain bridged by disulfide bonds); Fab' (e.g., an antibody fragment containing a single antigen binding domain, the antigen binding domain comprising Fab' and another portion of a heavy chain through a hinge region); F(ab')2 (e.g., two Fab' molecules linked by an interchain disulfide bond in the hinge region of the heavy chain; the Fab' molecules can be directed against the same or different epitopes); bispecific Fab (e.g., a Fab molecule with two antigen binding domains, each of which can be directed against a different epitope); a single chain containing a variable region, also called scFv (e.g., a single light chain and a variable antigen binding determining region of a heavy chain of an antibody linked together by a chain of 10-25 amino acids); disulfide-linked Fv or dsFv (e.g., a single light chain and a variable antigen binding determining region of a heavy chain of an antibody linked together by disulfide bonds); camelized VH (e.g., a variable antigen binding determining region of a single heavy chain of an antibody , in which some of the amino acids at the VH interface are those found in the heavy chain of a naturally occurring camelid antibody); bispecific scFv (e.g., an scFv or dsFv molecule having two antigen binding domains, each of which can be directed against a different epitope); dimeric antibodies (e.g., a dimeric scFv formed when the VH domain of a first scFv is assembled with the VL domain of a second scFv and the VL domain of the first scFv is assembled with the VH domain of a second scFv); the two antigen binding regions of a diabody can be directed against the same or different epitopes); triabodies (e.g., trimerized scFvs formed in a manner similar to diabodies, but in which three antigen binding domains are produced in a single complex; the three antigen binding domains can be directed against the same or different epitopes); and tetrabodies (e.g., tetrameric scFvs formed in a manner similar to diabodies, but in which four antigen binding domains are produced in a single complex; the four antigen binding domains can be directed against the same or different epitopes). In some aspects, the targeting moiety disclosed herein is an antibody or antigen-binding fragment thereof with monospecificity, bispecificity, trispecificity or multispecificity. In other aspects, the targeting moiety disclosed herein is a combination of one or more of the above antibodies or antigen-binding fragments thereof.

[0038] 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 conjugate disclosed herein is internalized into Schwann cells. In some instances, the ligand molecule comprises a peptide, a glycoprotein, a carbohydrate moiety, 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 neuromodulatory peptides. In some instances, the ligand molecule described herein binds to Cadm1. 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.

[0039] 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.

[0040] In some examples, the targeting moieties disclosed herein comprise at least a portion of neurofascin-186 (NF186) or a derivative thereof. In some examples, the targeting moieties disclosed herein comprise one or more neuromodulin binding domains of NF186.

[0041] In some instances, the targeting moiety disclosed herein comprises at least 1%, 5%, 10%, 20%, 30%, 40%, 50% or 60% of the full-length NF186 without significantly affecting the binding affinity to the neuromodulatory peptide. In some instances, the targeting moiety disclosed herein comprises no more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of the full-length NF186 without significantly affecting the binding affinity to the neuromodulatory peptide. In some instances, the targeting moiety disclosed herein comprises a NF186 portion having one or more conservative amino acid substitutions without significantly affecting the binding affinity to the neuromodulatory peptide. In some instances, the targeting moiety disclosed herein comprises a NF186 portion having one or more non-conservative amino acid substitutions without significantly affecting the binding affinity to the neuromodulatory peptide. In some instances, the targeting moiety disclosed herein comprises a portion of NF186 having one or more substitutions with basic, acidic, aromatic, aliphatic, amide, cyclic or sulfur-containing amino acids without significantly affecting the binding affinity to the neuromodulatory peptide. In some instances, the targeting moiety disclosed herein comprises a portion of NF186 having one or more substitutions with standard amino acids or non-standard amino acids without significantly affecting the binding affinity to the neuromodulatory peptide. In some instances, the targeting moiety disclosed herein comprises a portion of NF186 having one or more substitutions with α, β, γ or δ amino acids without significantly affecting the binding affinity to the neuromodulatory peptide. In some instances, the targeting moiety disclosed herein comprises a portion of NF186 having one or more substitutions with positively charged, negatively charged or neutral amino acids without significantly affecting the binding affinity to the neuromodulatory peptide. In some instances, the targeting moiety disclosed herein comprises a portion of NF186 having one or more substitutions with polar or non-polar amino acids without significantly affecting the binding affinity to the neuromodulatory peptide. In some instances, the targeting moiety disclosed herein comprises a portion of NF186 having one or more amino acid deletions without significantly affecting the binding affinity to the neuromodulatory peptide. In some instances, the targeting moiety disclosed herein comprises a portion of NF186 having one or more amino acid insertions without significantly affecting the binding affinity to the neuromodulatory peptide. In some instances, the targeting moiety disclosed herein comprises a portion of NF186 and an amino acid sequence that is at least partially opposite to the amino acid sequence of the corresponding portion of NF186 without significantly affecting the binding affinity to the neuromodulatory peptide. In some instances, the targeting moiety disclosed herein comprises a portion of NF186, and wherein at least one of the amino acids from the original portion of NF186 is replaced by a stereoisomer of the amino acid (e.g., a D-stereoisomer) without significantly affecting the binding affinity to the neuromodulatory peptide.In some examples, the targeting moiety disclosed herein comprises a portion of NF186, and one or more groups, such as D-amino acids, are extended at one or both ends thereof without significantly affecting the binding affinity to the neuromodulatory peptide. In some examples, the targeting moiety disclosed herein comprises NF186 with a combination of one or more of the above modifications without significantly affecting the binding affinity to the neuromodulatory peptide.

[0042] 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 neuromodulator 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 the full-length recombinant NF186 without significantly affecting the binding affinity with the neuromodulator. In some instances, the targeting moiety disclosed herein comprises no more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of the full-length recombinant NF186 without significantly affecting the binding affinity with the neuromodulator. In some instances, the targeting moiety disclosed herein comprises a recombinant NF186 portion having one or more conservative amino acid substitutions without significantly affecting the binding affinity with the neuromodulator. In some examples, the targeting moiety disclosed herein comprises a recombinant NF186 portion having one or more non-conservative amino acid substitutions without significantly affecting the binding affinity to the neuromodulatory peptide. In some examples, the targeting moiety disclosed herein comprises a portion of recombinant NF186 having one or more substitutions with basic, acidic, aromatic, aliphatic, amide, cyclic or sulfur-containing amino acids without significantly affecting the binding affinity to the neuromodulatory peptide. In some examples, the targeting moiety disclosed herein comprises a portion of recombinant NF186 having one or more substitutions with standard amino acids or non-standard amino acids without significantly affecting the binding affinity to the neuromodulatory peptide. In some examples, the targeting moiety disclosed herein comprises a portion of recombinant NF186 having one or more substitutions with α, β, γ or δ amino acids without significantly affecting the binding affinity to the neuromodulatory peptide. In some examples, the targeting moiety disclosed herein comprises a portion of recombinant NF186 having one or more substitutions with positively charged, negatively charged or neutral amino acids without significantly affecting the binding affinity to the neuromodulatory peptide. In some instances, the targeting moiety disclosed herein comprises a portion of recombinant NF186 having one or more substitutions with polar or non-polar amino acids without significantly affecting the binding affinity to the neuromodulatory peptide. In some instances, the targeting moiety disclosed herein comprises a portion of recombinant NF186 having one or more amino acid deletions without significantly affecting the binding affinity to the neuromodulatory peptide. In some instances, the targeting moiety disclosed herein comprises a portion of recombinant NF186 having one or more amino acid insertions without significantly affecting the binding affinity to the neuromodulatory peptide. In some instances, the targeting moiety disclosed herein comprises a portion of recombinant NF186 and an amino acid sequence that is at least partially opposite to the amino acid sequence of the corresponding portion of recombinant NF186 without significantly affecting the binding affinity to the neuromodulatory peptide.In some instances, the targeting moiety disclosed herein comprises a portion of recombinant NF186, and at least one of the amino acids from the original portion of the recombinant NF186 is replaced by a stereoisomer (e.g., D-stereoisomer) of the amino acid without significantly affecting the binding affinity to the neuromodulatory peptide. In some instances, the targeting moiety disclosed herein comprises a portion of recombinant NF186, and one or more groups, such as D-amino acids, are extended at one or both ends thereof without significantly affecting the binding affinity to the neuromodulatory peptide. In some instances, the targeting moiety disclosed herein comprises recombinant NF186 having a combination of one or more of the above modifications without significantly affecting the binding affinity to the neuromodulatory peptide.

[0043] In some instances, the targeting moiety disclosed herein binds to a nerve-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 α2-subunit of laminin 2. In some instances, the targeting moiety disclosed herein binds to the G domain of the α2-subunit of laminin 2. In some instances, the targeting moiety disclosed herein binds to the G1 domain of the α2-subunit of laminin 2. In some instances, the targeting moiety disclosed herein binds to the G2 domain of the α2-subunit of laminin 2. In some instances, the targeting moiety disclosed herein binds to the G3 domain of the α2-subunit of laminin 2. In some instances, the targeting moiety disclosed herein binds to the G4 domain of the α2-subunit of laminin 2. In some instances, the targeting moiety disclosed herein binds to the G5 domain of the α2-subunit of laminin 2. In some examples, the targeting moieties disclosed herein bind to the β1-subunit of laminin 2. In some examples, the targeting moieties disclosed herein bind to the γ1-subunit of laminin 2.

[0044] In some examples, the targeting moiety disclosed herein comprises at least a portion of the glycolipid PGL-1 or a derivative thereof. In some examples, the targeting moiety disclosed herein comprises a unique trisaccharide of PGL-1 (3,6-di-O-methylglucose linked α-1→4 to 2,3-di-O-methylrhamnose linked β-1→2 to 3-O-methylrhamnose).

[0045] In some instances, the targeting moiety disclosed herein comprises at least a portion of ML-LBP21 (also referred to as histone-like protein / Hlp) or a derivative thereof. In some instances, the targeting moiety disclosed herein comprises the 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 the full-length ML-LBP21 without significantly affecting the binding affinity to laminin-2. In some instances, the targeting moiety disclosed herein comprises no more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of the 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 having 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 having 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 ML-LBP21 having one or more substitutions with basic, acidic, aromatic, aliphatic, amide, cyclic or sulfur-containing amino acids, without significantly affecting the binding affinity to laminin-2. In some instances, the targeting moiety disclosed herein comprises a portion of ML-LBP21 having one or more substitutions with standard amino acids or non-standard amino acids, without significantly affecting the binding affinity to laminin-2. In some instances, the targeting moiety disclosed herein comprises a portion of ML-LBP21 having one or more substitutions with α, β, γ or δ amino acids, without significantly affecting the binding affinity to laminin-2. In some instances, the targeting moiety disclosed herein comprises a portion of ML-LBP21 having one or more substitutions with positively charged, negatively charged or neutral amino acids without significantly affecting the binding affinity to laminin-2. In some instances, the targeting moiety disclosed herein comprises a portion of ML-LBP21 having one or more substitutions with polar or non-polar amino acids without significantly affecting the binding affinity to laminin-2. In some instances, the targeting moiety disclosed herein comprises a portion of ML-LBP21 having 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 having 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 that is at least partially opposite to 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 at least one of the amino acids from the original portion of ML-LBP21 is replaced by a stereoisomer (e.g., D-stereoisomer) of the 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 one or more groups, such as D-amino acids, are extended at one or both ends thereof, 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 modifications, without significantly affecting the binding affinity to laminin-2.

[0046] In some examples, the targeting moiety disclosed herein comprises a recombinant ML-LBP21 peptide or a derivative thereof. In some examples, the targeting moiety disclosed herein comprises a laminin-2 binding domain of recombinant ML-LBP21. In some examples, the targeting moiety disclosed herein comprises at least 1%, 5%, 10%, 20%, 30%, 40%, 50% or 60% of the full-length recombinant ML-LBP21 without significantly affecting the binding affinity to laminin-2. In some examples, the targeting moiety disclosed herein comprises no more than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of the full-length recombinant ML-LBP21 without significantly affecting the binding affinity to laminin-2. In some examples, the targeting moiety disclosed herein comprises a portion of the full-length 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 having 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 full-length ML-LBP21 having one or more substitutions with basic, acidic, aromatic, aliphatic, amide, cyclic or sulfur-containing amino acids without significantly affecting the binding affinity to laminin-2. In some instances, the targeting moiety disclosed herein comprises a portion of recombinant ML-LBP21 having one or more substitutions with standard amino acids or non-standard amino acids without significantly affecting the binding affinity to laminin-2. In some instances, the targeting moiety disclosed herein comprises a portion of recombinant ML-LBP21 having one or more substitutions with α, β, γ or δ amino acids without significantly affecting the binding affinity to laminin-2. In some instances, the targeting moiety disclosed herein comprises a portion of recombinant ML-LBP21 having one or more substitutions with positively charged, negatively charged or neutral amino acids without significantly affecting the binding affinity to laminin-2. In some instances, the targeting moiety disclosed herein comprises a portion of recombinant ML-LBP21 having one or more substitutions with polar or non-polar amino acids without significantly affecting the binding affinity to laminin-2. In some instances, the targeting moiety disclosed herein comprises a portion of ML-LBP21 having 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 having 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 that is at least partially opposite to 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 at least one of the amino acids from the original portion of the recombinant ML-LBP21 is replaced by a stereoisomer (e.g., D-stereoisomer) of the 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 one or more groups, such as D-amino acids, are extended at one or both ends thereof, without significantly affecting the binding affinity to laminin-2. In some instances, the targeting moiety disclosed herein comprises a recombinant ML-LBP21 having a combination of one or more of the above modifications, without significantly affecting the binding affinity to laminin-2.

[0047] In some aspects, the targeting moieties disclosed herein comprise a combination of one or more of the above-described molecules.

[0048] Drug molecules

[0049] In some aspects, the drug molecules disclosed herein modify the expression of disease-related molecules. In specific aspects, the overexpression of disease-related molecules causes disease. Therefore, in some instances, the drug molecules disclosed herein reduce the transcription / RNA levels of disease-related molecules by at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%. Therefore, in some instances, the drug molecules disclosed herein reduce the transcription / RNA levels of disease-related molecules to the level of healthy individuals. Therefore, in other examples, the drug molecules disclosed herein reduce the translation / protein level of disease-related molecules by at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%. Therefore, in some examples, the drug molecules disclosed herein reduce the translation / protein level of disease-related molecules to the level of healthy individuals. In a specific aspect, the lack of disease-related molecules causes the disease. Therefore, in some examples, the drug molecules disclosed herein increase the transcription / RNA level of disease-related molecules by at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%. Therefore, in some instances, the drug molecules disclosed herein increase the transcription / RNA level of disease-related molecules to the level of healthy individuals. Therefore, in other instances, the drug molecules disclosed herein increase the translation / protein level of disease-related molecules by at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%. Therefore, in some instances, the drug molecules disclosed herein increase the translation / protein level of disease-related molecules to the level of healthy individuals.

[0050] In other aspects, the drug molecules disclosed herein modify the activity of disease-related molecules. In specific aspects, the overactivity of disease-related molecules causes disease. Therefore, in some instances, the drug molecules disclosed herein reduce the activity of disease-related molecules by at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%. Therefore, in some instances, the drug molecules disclosed herein reduce the activity of disease-related molecules to the level of healthy individuals. In specific aspects, the low activity of disease-related molecules causes disease. Therefore, in some instances, the drug molecules disclosed herein increase the activity of disease-related molecules by at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%. Therefore, in some instances, the drug molecules disclosed herein increase the activity of disease-related molecules to the level of healthy individuals.

[0051] In some instances, the drug molecule comprises a precursor form of a drug or drug (drug precursor or prodrug). In some instances, the drug molecule comprises a peptide. In some instances, the drug molecule comprises a protein. As described herein, any suitable peptide or protein can be used as a drug molecule to modify the expression or activity of a disease-related molecule. In some instances, the drug molecule comprises an enzyme. In some instances, several methods can be used to produce, synthesize and / or derive peptides or proteins, such as phage display peptide libraries, single bead single compound peptide libraries, directed evolution, or position scanning synthetic peptide combinatorial libraries. In some instances, the drug molecule comprises a 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, such as cysteine, alanine, or non-naturally occurring or modified amino acids. Non-naturally occurring amino acids include 3-amino acids, high-amino acids, proline derivatives, 3-substituted alanine derivatives, linear core amino acids, N-methyl amino acids, and other amino acids known in the art. In some instances, the peptide can be linear; in other instances, the peptide can be cyclic, such as a bicyclic.

[0052] In some instances, the drug molecule comprises a small molecule. In some instances, the small molecule is a modulator of a disease-related molecule. In some instances, the small molecule is an agonist of a disease-related molecule. In some instances, the small molecule is an antagonist of a disease-related molecule. In some instances, the small molecule is an inhibitor of a disease-related molecule.

[0053] In some instances, the drug molecule comprises an oligonucleotide. In some instances, the oligonucleotide comprises a double-stranded RNAi molecule or a single-stranded antisense oligonucleotide. In a particular instance, 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 a precursor thereof. In other instances, the drug molecule comprises a nucleic acid described in Sandy et al., Mammalian RNAi: a practical guide, BioTechniques, VOL. 39, NO. 2 REVIEW, which is incorporated herein by reference in its entirety.

[0054] In some instances, the gene expression construct encodes a gene editing enzyme. In some instances, the drug molecule comprises a CRISPR-based tool, a meganuclease-based tool, a zinc finger nuclease (ZFN)-based tool, or a transcription activator-like effector nuclease (TALEN)-based tool.

[0055] In some instances, the length of the double-stranded RNAi molecule or the single-stranded antisense oligonucleotide is about 9 to about 40 nucleotides. In some instances, the length of the double-stranded RNAi molecule or the single-stranded antisense oligonucleotide is about 9 to about 36 nucleotides. In some instances, the length of the double-stranded RNAi molecule or the single-stranded antisense oligonucleotide is about 10 to about 30 nucleotides. In some instances, the length of the double-stranded RNAi molecule or the single-stranded antisense oligonucleotide is about 15 to about 30 nucleotides. In some instances, the length of the double-stranded RNAi molecule or the single-stranded antisense oligonucleotide is about 20 to about 30 nucleotides. In some instances, the length of the double-stranded RNAi molecule or the single-stranded antisense oligonucleotide is about 10 to about 25 nucleotides. In some instances, the length of the double-stranded RNAi molecule or the single-stranded antisense oligonucleotide is about 10 to about 20 nucleotides. In some instances, the length of the double-stranded RNAi molecule or the single-stranded antisense oligonucleotide is at least 5, 6, 7, 8, 9, 10, 15, 20, 25 or 30 nucleotides. In some instances, the length of the double-stranded RNAi molecule or single-stranded antisense oligonucleotide is no more than 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 nucleotides. In some instances, the length of the double-stranded RNAi molecule or single-stranded antisense oligonucleotide is about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides.

[0056] In some instances, drug molecules disclosed herein are double-stranded RNAi molecules with sense strands and antisense strands. In some aspects, the sense strands and antisense strands described herein are reverse complementary to each other and form a duplex with a 3' overhang on the antisense strand. In some aspects, the sense strands and antisense strands described herein are reverse complementary to each other and form a duplex with a 5' overhang on the antisense strand. In some aspects, the sense strands and antisense strands described herein are reverse complementary to each other and form a duplex with a 3' overhang on the sense strand. In some aspects, the sense strands and antisense strands described herein are reverse complementary to each other and form a duplex with a 5' overhang on the sense strand.

[0057] In some examples, the double-stranded RNAi molecule or single-stranded antisense oligonucleotide comprises at least one modified 2' modified nucleotide. In some examples, the double-stranded RNAi molecule or single-stranded antisense oligonucleotide comprises at least one modified internucleotide bond.

[0058] In some instances, the double-stranded RNAi molecules or single-stranded antisense oligonucleotides described herein include one or more sugar-modified nucleotides. In some specific examples, the sugar-modified nucleotides are 2'-fluoro-modified nucleotides. In some specific examples, the sugar-modified nucleotides are 2'-alkoxy-modified nucleotides (e.g., 2'-methoxy-modified nucleotides). In some specific examples, the sugar-modified nucleotides are 2'-amino-modified nucleotides. In some specific examples, the sugar-modified nucleotides are 2'-azido-modified nucleotides.

[0059] In some instances, double-stranded RNAi molecules or single-stranded antisense oligonucleotides as described herein comprise nucleotides modified by one or more main chains. In some instantiations, the modified main chain is methylphosphonate. In some instantiations, the modified main chain is phosphorothioate. In some instantiations, the modified main chain is guanidinopropylphosphoramidate. In some instantiations, the modified main chain is mesyl-phosphoramidate (MsPA) key. In some instantiations, the modified main chain is phosphorothioate, and phosphorothioate is the phosphorothioate of stereochemical enrichment.

[0060] In some instances, the double-stranded RNAi molecules or single-stranded antisense oligonucleotides described herein comprise one or more purine modifications. In some specific instances, the purine modifications described herein are 2,6-diaminopurine. In some specific instances, the purine modifications described herein are 3-deaza-adenine. In some specific instances, the purine modifications described herein are 7-deaza-guanine. In some specific instances, the purine modifications described herein are 8-azido-adenine.

[0061] In some instances, the double-stranded RNAi molecules or single-stranded antisense oligonucleotides described herein comprise one or more pyrimidine modifications. In some specific instances, the pyrimidine modifications described herein are 2-thio-thymidine. In some specific instances, the pyrimidine modifications described herein are 5-formamide-uracil. In some specific instances, the pyrimidine modifications described herein are 5-methyl-cytosine. In some specific instances, the pyrimidine modifications described herein are 5-ethynyluracil.

[0062] In some instances, polynucleic acid molecules described herein include abasic substitutions. In those instances where a heterozygous polynucleotide construct is considered to be used as siRNA, it is desirable to reduce the off-target effect of miRNA samples. Including one or more (e.g., one or two) abasic substitutions in a heterozygous polynucleotide construct can reduce or even eliminate the off-target effect of miRNA samples, because abasic substitutions lack the core bases that can participate in base pairing interactions and alleviate steric hindrance. Therefore, double-stranded RNAi molecules or single-stranded antisense oligonucleotides disclosed herein may include one or more (e.g., one or two) abasic substitutions. In a specific example, abasic substitutions are at the 5th nucleotide at the end of the antisense strand 5' described herein. Polynucleotide molecules described herein may contain a chain including a seed region, and the seed region includes nucleosides (e.g., inosine) containing hypoxanthine core bases.

[0063] In some examples, double-stranded RNAi molecules or single-stranded antisense oligonucleotides described herein include one or more types of modifications as described above. Therefore, in some examples, about 10% of the nucleotides from double-stranded RNAi molecules or single-stranded antisense oligonucleotides described herein are modified with one or more types of modifications as described above. In other examples, about 20% of the nucleotides from double-stranded RNAi molecules or single-stranded antisense oligonucleotides described herein are modified with one or more types of modifications as described above. In other examples, about 30% of the nucleotides from double-stranded RNAi molecules or single-stranded antisense oligonucleotides described herein are modified with one or more types of modifications as described above. In other examples, about 40% of the nucleotides from double-stranded RNAi molecules or single-stranded antisense oligonucleotides described herein are modified with one or more types of modifications as described above. In other examples, about 50% of the nucleotides from double-stranded RNAi molecules or single-stranded antisense oligonucleotides described herein are modified with one or more types of modifications as described above. In other examples, about 60% of the nucleotides from double-stranded RNAi molecules or single-stranded antisense oligonucleotides described herein are modified with one or more types of modifications as described above. In other examples, about 70% of the nucleotides from double-stranded RNAi molecules as described herein or single-stranded antisense oligonucleotides are modified with one or more types of modifications as described above. In other examples, about 80% of the nucleotides from double-stranded RNAi molecules as described herein or single-stranded antisense oligonucleotides are modified with one or more types of modifications as described above. In other examples, about 90% of the nucleotides from double-stranded RNAi molecules as described herein or single-stranded antisense oligonucleotides are modified with one or more types of modifications as described above. In other examples, 100% of the nucleotides from double-stranded RNAi molecules as described herein or single-stranded antisense oligonucleotides are modified with one or more types of modifications as described above.

[0064] In some instances, one or more types of modification described herein occur at different positions in double-stranded RNAi molecules or single-stranded antisense oligonucleotides as described herein. In a specific instance, one or more types of modification described herein occur in the seed region in double-stranded RNAi molecules or single-stranded antisense oligonucleotides as described herein. In a specific instance, one or more types of modification described herein occur at the 3' end of double-stranded RNAi molecules or single-stranded antisense oligonucleotides as described herein. In a specific instance, one or more types of modification described herein occur at the 5' end of double-stranded RNAi molecules or single-stranded antisense oligonucleotides as described herein. In a specific instance, one or more types of modification described herein occur in double-stranded RNAi molecules or single-stranded antisense oligonucleotides as described herein. In a specific instance, one or more types of modification described herein occur in clusters in double-stranded RNAi molecules or single-stranded antisense oligonucleotides as described herein.

[0065] In some instances, the disease-related molecule is peripheral myelin protein 22 (PMP22). Therefore, the drug molecules disclosed herein modify PMP22. In some specific instances, PMP22 is associated with Charcot-Marie-Tooth disease type 1A (CMT1A). CMT damages the peripheral nervous system, and different types of CMT lead to impaired electrical information to muscles and their subsequent malfunction. Specifically, CMT1A, the most common type of CMT (55%-60% of total CMT patients), usually begins in adolescence, accompanied by muscle weakness and atrophy of the lower legs, followed by hand weakness and reduced tactile sensation (e.g., touch, heat, cold) in the later stages of life. The disease is a progressive disease, and its symptoms usually worsen after the age of 45. The symptoms of worsening include a decreased ability to perform simple tasks, unstable walking, and a tendency to be injured by multiple falls, as well as a decreased quality of life. The genetic defect in most CMT1A patients is a 1.5Mb duplication on a chromosome segment containing the PMP22 gene. The increase in PMP22 protein over the basal concentration leads to demyelination.

[0066] In some examples, the double-stranded RNAi molecule or single-stranded antisense oligonucleotide comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, 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% complementary to the target region of the human PMP22 transcript. In other examples, the double-stranded RNAi molecule or single-stranded antisense oligonucleotide comprises a contiguous sequence of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length that is complementary to the target region of the human PMP22 transcript with no more than 1, 2, 3, 4 or 5 mismatches. In other examples, the double-stranded RNAi molecule or single-stranded antisense oligonucleotide comprises a continuous sequence of about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length, which is complementary to the target region of the human PMP22 transcript with no more than 1, 2, 3, 4 or 5 mismatches. In some examples, the target region of the human PMP22 transcript is in the 5'UTR of the human PMP22 transcript. In some examples, the target region of the human PMP22 transcript is in the 3'UTR of the human PMP22 transcript. In some examples, the target region of the human PMP22 transcript is in the coding region of the human PMP22 transcript. In some examples, the target region of the human PMP22 transcript is in exon 1 of the human PMP22 transcript. In some examples, the target region of the human PMP22 transcript is in exon 2 of the human PMP22 transcript. In some examples, the target region of the human PMP22 transcript is in exon 3 of the human PMP22 transcript. In some examples, the target region of the human PMP22 transcript is in exon 4 of the human PMP22 transcript. In some examples, the target region of the human PMP22 transcript is in exon 5 of the human PMP22 transcript. In some examples, the target region of the human PMP22 transcript is in exon 6 of the human PMP22 transcript. In some examples, the target region of the human PMP22 transcript is in exon 7 of the human PMP22 transcript. In some examples, the target region of the human PMP22 transcript includes an intron portion of the pre-PMP22 transcript. In some examples, the target region of the human PMP22 transcript includes an exon-intron junction of the pre-PMP22 transcript.

[0067] In some instances, the disease-associated molecule is an anti-ganglioside antibody. Therefore, the drug molecules disclosed herein modify the anti-ganglioside antibody. In some instances, the anti-ganglioside antibody is related to Guillain-Barré syndrome. Guillain-Barré syndrome is a post-infectious immune-mediated neuropathy caused by autoimmune destruction of nerves in the peripheral nervous system. The syndrome can include muscle movements and movements that transmit pain, temperature, and touch. This may lead to muscle weakness and loss of sensation in the legs and / or arms. Anti-ganglioside antibodies may be related to the pathogenesis of Guillain-Barré syndrome.

[0068] In some instances, the disease-associated molecule is a subunit of the SWI / SNF protein complex, which is encoded by the gene SMARCB1. In some instances, the disease-associated molecule is the LZTR1 protein, which is encoded by the LZTR1 gene. Therefore, the drug molecules disclosed herein modify the subunits of the SWI / SNF protein complex. Therefore, the drug molecules disclosed herein modify the LZTR1 protein. In some instances, the subunits of the SWI / SNF protein complex are associated with schwannomatosis. In some instances, the LZTR1 protein is associated with schwannomatosis. Schwannomatosis is a neurofibromatosis that causes multiple nerve sheath tumors.

[0069] In some instances, the drug molecule is an immunoglobulin, and the associated disease is chronic inflammatory demyelinating polyneuropathy (CIDP). CIDP is a rare peripheral neuropathy 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 for months or even years, resulting in significant nerve damage that can limit and delay response to treatment. Most individuals may require long-term treatment; without treatment, almost one-third of CIDP patients may develop wheelchair dependence. Early identification and appropriate treatment are crucial to helping patients avoid substantial disability.

[0070] Coupling of target moiety and drug molecule

[0071] In certain examples, the targeting moieties disclosed herein can be coupled or conjugated to the drug molecules disclosed herein directly (e.g., by 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.

[0072] In some instances, the targeting moiety disclosed herein and the drug molecule disclosed herein are connected by non-covalent interactions. In a particular instance, the non-covalent interaction comprises biotin / avidin interactions. The example of connecting the connection of the targeting moiety and the drug molecule includes but is not limited to using biotin and avidin or streptavidin (see, e.g., U.S. Patent No. 4,885,172A), typical biotin / avidin substitutes (e.g. Harmer and Samuel (1989) J. Immunol. Meth. 122 (1): 115-221), dioxigenin / anti-dioxigenin, etc.

[0073] In other examples, the targeting moiety disclosed herein and the drug molecule disclosed herein are connected by covalent interactions. In a particular example, the targeting moiety disclosed herein and the drug molecule disclosed herein are connected by chemical conjugation. Traditional chemical conjugation using, for example, a bifunctional coupling agent, such as glutaraldehyde, diimide esters, aromatic and aliphatic diisocyanates, dicarboxylic acid bis-p-nitrophenyl esters, aromatic disulfonyl chlorides and bifunctional aryl halides, such as 1,5-difluoro-2,4-dinitrobenzene; p, p'-difluoro m, m'-dinitrodiphenyl sulfone, sulfhydryl reactive maleimide, etc.

[0074] In some examples, the chemical conjugation of a targeting moiety disclosed herein and a drug molecule disclosed herein is via a non-cleavable linker or via a cleavable linker.

[0075] In some instances, the cleavable linker is generally cleavable only within the cell and is preferably stable in the extracellular environment outside the cell, such as Schwann cells. In a specific instance, the cleavable linker disclosed herein comprises a chemically cleavable linker. In a specific instance, the cleavable linker disclosed herein comprises an enzymatically cleavable linker.

[0076] Many different chemically cleavable linkers are known to those skilled in the art (see, e.g., U.S. Pat. Nos. 4,618,492; 4,542,225 and 4,625,014). In some specific examples, the chemically cleavable linker is a pH-sensitive linker. In some examples, the pH-sensitive linker disclosed herein is easily degraded in a high pH environment. In some examples, the pH-sensitive linker disclosed herein is easily degraded in a low pH environment. In some examples, the pH-sensitive linker can be cut at a pH in the range of 4 to 6. In some examples, the pH-sensitive linker comprises a hydrazone or a cyclic acetal. In some examples, the pH-sensitive linker is cut in an endosome or lysosome. In other specific examples, the chemically cleavable linker is a glutathione-sensitive linker. In some examples, the glutathione-sensitive linker is cut by a disulfide exchange reaction with a glutathione species in the cell. In some examples, the disulfide portion further comprises at least one amino acid, such as a cysteine ​​residue. Other exemplary chemically cleavable linkers include, but are not limited to, acid-labile linkers, disulfide linkers, and the like. Acid labile joints are designed to be stable at pH levels encountered in blood, but become unstable and degraded when encountering low pH environments in lysosomes. Acid labile joints, comprising a portion selected from the group consisting of: hydrazones, acetals, cis-aconitamides and silyl ethers. Acid-sensitive joints 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 a plasma half-life of 4.4 hours at pH 5, indicating that they are selectively cleavable under acidic conditions such as those found in lysosomes, (see, e.g., Doronina et al. 92013) Nat. Biotechnol. 21 (7): 778-784). The disulfide bridge is a cleavable linker that takes 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, the disulfide bond can release the drug in the lysosome.

[0077] The enzymatically cleavable linker is selected to be cleaved by an enzyme (e.g., a protease). In some examples, the protease cleavable linker is generally designed to be stable in blood / plasma, but rapidly cleaved by lysosomal enzymes in lysosomes. The most popular enzyme cleavage sequence is the dipeptide valine-citrulline, combined with the self-immolative linker p-aminobenzyl alcohol (PAB). Cleavage of the amide-linked PAB triggers 1,6-elimination of carbon dioxide and concomitant release of the free drug in the form of the parent amine (see, e.g., Burke et al. (2009) Bioconjug. Chem. 20(6): 1242-1250). In some specific examples, the cleavable linker is a protease sensitive linker. The protease sensitive linker described herein may comprise a sequence that can be cleaved by a lysosomal protease and / or an endosomal protease. In some examples, the protease sensitive linker generally comprises a peptide sequence 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 a naturally occurring amino acid, such as cysteine, alanine or a non-naturally occurring or modified amino acid. Non-naturally occurring amino acids include 3-amino acids, high-amino acids, proline derivatives, 3-substituted alanine derivatives, linear core amino acids, N-methyl amino acids. In some instances, the protease sensitive linker comprises a valine-citrulline or alanine-citrulline dipeptide sequence. In some instances, the protease sensitive linker can be cleaved by lysosomal proteases, such as cathepsin B and / or endosomal proteases.

[0078] In some examples, the targeting moiety and the drug molecule are covalently linked to each other via a non-cleavable linker. Typically, non-cleavable links are not easily degraded in cells or physiological environments. In some examples, the non-cleavable linker described herein comprises an optionally substituted alkyl, wherein the substitution may include halogen, hydroxyl, oxygen species, and other common substitutions. In some examples, 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 polysaccharide, a sugar that cannot be degraded by an enzyme, an azide, an alkyne azide, a peptide sequence comprising an LPXT sequence, a thioether, biotin, a biphenyl, a repeating unit of polyethylene glycol or an equivalent compound, an acid ester, an acid amide, a sulfonamide, and / or an alkoxyamine linker. In some examples, a drug molecule comprising an LPXT sequence is covalently linked to a targeting moiety comprising (G) using a sortase-mediated connection. In other examples, a targeting moiety comprising an LPXT sequence is covalently linked to a drug molecule comprising a (G) sequence using sortase-mediated ligation (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, the entire contents of which are incorporated herein by reference). In some examples, the non-cleavable linker described herein comprises a 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, the 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 group, an optionally substituted nitrogen species, an optionally substituted oxygen species O, an optionally substituted sulfur species, or a poly(oxyalkylene), such as polyethylene oxide or polypropylene oxide.

[0079] In some instances, the linker described herein is a peptide linker. In various instances, the peptide linker is relatively short, generally 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 a single amino acid. In some instances, the peptide linker disclosed herein includes an amino acid sequence that can be cut by a protease. In some instances, the peptide linker disclosed herein includes an amino acid sequence that can be cut by a cathepsin. In some instances, the peptide linker disclosed herein includes a dipeptide valine-citrulline (Val-Cit) (see U.S. Patent No. 6,214,345, the entire contents of which are incorporated herein by reference) or Phe-Lys. A dipeptide linker library was screened by Debowchik and colleagues 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 sharp contrast, Val-Cit had a half-life of 240 min. They also found that removal of the PAB group reduced the cleavage rate, presumably by steric interference with enzyme binding. Another study compared the potency of the auristatin derivative MMAE linked by the dipeptide linkers Phe-Lys and Val-Cit and a similar hydrazone linker. The stability of the Val-Cit linker in human plasma has been shown to be more than 100 times that of the hydrazone linker. Most importantly, the stability of the Phe-Lys linker in human plasma is significantly lower than that of Val-Cit, which explains its current popularity (see, e.g., Doronina et al. (2003) Nat. Biotechnol. 21(7):778-784).

[0080] Other peptide linkers include, but are 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, GGGGS GGGGS GGGGS GGGGS, GGGGS GGGGS GGGGS GGGGS GGGGS, GGGGS GGGGS GGGGS GGGGS GGGGS, GGGGS GGGGS GGGGS GGGGS FK GGGGSGGGGS GGGGS, and GGGGS GGGGS GGGGS VA GGGGS GGGGS GGGGS.

[0081] In some examples, the linkers described herein are utilized Technology, which is a genetic fusion with a conformationally disordered polypeptide sequence consisting of amino acids Pro, Ala and / or Ser. In other examples, the linker described herein is selected from patents such as the linker disclosed in U.S. Patent No. 10,961,287, the entire contents of which are incorporated herein by reference.

[0082] In some examples, the linker described herein is a non-peptide linker. Glucuronide linkers incorporate hydrophilic sugar groups that are cleaved by the lysosomal enzyme β-glucuronidase. Once the sugar is cleaved from the phenolic backbone, the self-decomposition of the PAB group releases the conjugated portion (see, e.g., Jeffrey et al. (2006) Bioconjug. Chem. 17 (3): 831-840). In some examples, the linker described herein is derived from a natural multi-domain protein (see, e.g., Chen at al., (2013) Adv Drug Deliv Rev. 65 (10): 1357–1369).

[0083] The targeting moiety disclosed herein can be connected to the drug molecule disclosed herein via certain functional groups or via certain reactions. In some instances, the linker disclosed herein comprises a functional group that reacts with the corresponding functional group on the targeting moiety disclosed herein and / or the drug molecule disclosed herein. The bifunctional linker has a functional group that reacts with a group on the targeting moiety disclosed herein and another functional group that reacts with the drug molecule disclosed herein, and can be used to form a desired conjugate. In some instances, the heterobifunctional linker comprises two or more different reactive groups that react with the sites on the targeting moiety disclosed herein and the drug molecule disclosed herein, respectively. For example, a heterobifunctional cross-linker such as cysteine ​​may include an amine reactive group, and a thiol reactive group may interact with an aldehyde on a derivatized peptide. Other combinations of reactive groups suitable for heterobifunctional cross-linkers include, for example, amine reactive groups 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 reaction and functional group are illustrative and non-restrictive.Other exemplary suitable reactive groups include but are not limited to mercaptan (-SH), carboxylate (COOH), carboxyl (-COOH), carbonyl, amine (NH2), hydroxyl (-OH), aldehyde (-CHO), alcohol (ROH), ketone (R2CO), active hydrogen, ester, sulfydryl (SH), phosphate (-PO3) or photoreactive part.Amine reactive groups include but are not limited to such as isothiocyanate, isocyanate, acyl azide, NHS ester, sulfonyl chloride, aldehyde and glyoxal, epoxide and oxirane, carbonate, arylating agent, acylide ester, carbodiimide and anhydride.Thiol reactive groups include but are not limited to such as haloacetyl and alkyl halide derivatives, maleimide, aziridine, acryloyl derivatives, arylating agent and mercaptan-disulfide exchange reagent.Carboxylate reactive groups include but are not limited to such as diazoalkane and diazoacetyl compound, such as carbonyl diimidazole and carbodiimide. Hydroxyl reactive groups include, but are not limited to, epoxides and oxiranes, carbonyl diimidazoles, oxidation with periodate, N,N′-disuccinimidyl carbonate or N-hydroxysuccinamido chloroformate, enzyme oxidation, alkyl halides and isocyanates. Aldehyde and ketone reactive groups include, but are not limited to, hydrazine derivatives such as for Schiff base formation or reductive amination. Active hydrogen reactive groups include, but are not limited to, diazo derivatives such as for Mannich condensation and iodination reactions. Photoreactive groups include, but are not limited to, aryl azides and halogenated aryl azides, benzophenones, diazo compounds and diazirine derivatives.

[0084] In some instances, the linker described herein is connected to the targeting moiety by the cycloaddition reaction between azide and alkyne to form triazole, wherein azide and alkyne can be located on the targeting moiety, drug molecule or linker. In some instances, alkyne can be a cyclic alkyne, such as cyclooctyne. In some instances, alkyne can be a bicyclic nonyne (also referred to as bicyclic [6.1.0] nonyne or BCN) or a substituted bicyclic nonyne. In some instances, cyclooctane is as described in the international patent application publication WO2011136645 entitled "Fused Cyclooctyne Compounds And Their Use In Metal free Click Reactions (fused cyclooctyne compounds and their use in metal-free click reactions)" published on November 3, 2011. In some instances, azide can be a sugar or carbohydrate molecule comprising azide. In some instances, azide can be 6-azido-6-deoxygalactose or 6-azido-N-acetylgalactosamine. In some examples, the sugar or carbohydrate molecule comprising an azide is as described in International Patent Application Publication No. WO2016170186, published on October 27, 2016, entitled “Process For The Modification Of A Glycoprotein Using A Glycosyltransferase That Is Or Is Derived From A β(1,4)-N-Acetylgalactosaminyltransferase”.In some examples, a cycloaddition reaction is performed between an azide and an alkyne to form a triazole, wherein the azide and the alkyne can be located on a targeting moiety, a drug molecule, or a linker, as described in International Patent Application Publication No. WO2014065661, entitled “Modified antibody, antibody-conjugate and process for the preparation thereof”, published on May 1, 2014; or International Patent Application Publication No. WO2016170186, entitled “Process For The Modification Of A Glycoprotein Using A Glycosyltransferase That Is Or IsDerived From Aβ(1,4)-N-Acetylgalactosarninyltransferase”, published on October 27, 2016.

[0085] In some instances, joint as herein described is connected to the targeting moiety of drug molecule by the Diels-Alder reaction between dienophile and diene / heterodiene, wherein dienophile and alkene / heterodiene can be located on targeting moiety, drug molecule or connector. In some instances, joint is connected with targeting moiety and / or drug molecule by other pericyclic reactions such as alkene reaction. In some instances, joint disclosed herein is connected to targeting moiety and / or drug molecule by amide, thioamide or sulfonamide bond reaction. In some instances, joint disclosed herein is connected to the targeting moiety of drug molecule by condensation reaction, to form oxime, hydrazone or semicarbazide group present between joint, targeting moiety and drug molecule.

[0086] In some instances, the joint disclosed herein is connected to the targeting moiety by the conjugate addition reaction between the nucleophilic reagent of for example amine or hydroxyl and the electrophilic reagent of for example carboxylic acid or aldehyde.In some instances, before the reaction between the joint and the targeting moiety or drug molecule, the nucleophilic reagent may be present on the joint, and the electrophilic reagent may be present on the targeting moiety or drug molecule.In some instances, before the reaction between the joint and the targeting moiety or drug molecule, the electrophilic reagent may be present on the joint, and the nucleophilic reagent may be present on the targeting moiety or drug molecule.In some instances, the electrophile can be azide, silicon center, carbonyl, carboxylic acid, anhydride, isocyanate, thioisocyanate, succinimide ester, sulfosuccinimide ester, maleimide, alkyl halide, alkyl pseudohalide, epoxide, episulfide, aziridine, aryl, activated phosphorus center and / or activated sulfur center. In some examples, the nucleophile can be an optionally substituted alkene, an optionally substituted alkyne, an optionally substituted aryl, an optionally substituted heterocyclyl, a hydroxyl, an amino, an alkylamino, an anilino, or a thiol group.

[0087] In some examples, the linkers described herein further comprise a spacer, such as a polyethylene glycol spacer or an acyl / carbamoylsulfonamide spacer, such as a HydraSpace TM Spacer. In some examples, the spacer is as described in Verkade, JMM et al., "A Polar Sulfamide Spacer Significantly Enhances the Manufacturability, Stability, and Therapeutic Index of Antibody-Drug Conjugates", Antibodies, 2018, 7, 12, the entire contents of which are incorporated herein by reference.

[0088] Typically, the joints disclosed herein have no specific biological activity except for connecting proteins or maintaining some minimum distance or other spatial relationship between them. However, the constituent amino acids of the spacer can be selected to affect some properties of the molecule, such as folding, net charge or hydrophobicity. In some instances, the joint may include an enzyme cleavage site.

[0089] After chemical synthesis, biological expression or purification, the fusion protein may have a conformation that is substantially different from the native conformation of the constituent polypeptides. In this example, it may be necessary to denature and reduce the polypeptide and then refold the polypeptide into a preferred conformation. Methods for reducing and denaturing proteins and inducing refolding are well known to those skilled 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).

[0090] In some instances, one of the targeting moiety and the drug molecule is a peptide / protein or an antibody or its antigen-binding fragment, and the other is a nucleotide. In some instances, the peptide / protein or its antibody or antigen-binding fragment comprises a non-natural amino acid to which the nucleotide can be covalently linked. In some instances, the peptide / protein or its antibody or antigen-binding fragment is covalently linked to the nucleotide via a lysine residue or a cysteine ​​residue conjugated to the peptide / protein or its antibody or antigen-binding fragment. In some instances, the nucleotide is conjugated to the cysteine ​​residue of the peptide / protein or antibody or its antigen-binding fragment via a maleimide-containing joint, and optionally wherein the maleimide-containing joint comprises a maleimidocaproyl or maleimidomethylcyclohexane-1-carboxylate group. In some instances, the peptide / protein or antibody or its antigen-binding fragment is glycosylated, comprising at least one sugar moiety covalently linked to the nucleotide. In some instances, at least one sugar moiety comprises at least one sugar moiety that is a branched mannose. In some instances, the peptide / protein or its antibody or antigen binding fragment is glycosylated, comprising more than one sugar moiety, and each of all or part of more than one sugar moiety is covalently linked to a separate nucleotide. In some instances, the peptide / protein or its antibody or antigen binding fragment is fully glycosylated or partially glycosylated. Partial glycosylation can be produced by chemical or enzymatic methods. In some instances, partial glycosylation is produced in cells lacking enzymes in N- or O-glycosylation pathways. In some instances, the linker described herein is connected to the peptide / protein or antibody or its antigen binding fragment by a phosphate, thioether, ether, carbon-carbon or amide bond. In some instances, the linker described herein is connected to the nucleotide by a phosphate or phosphorothioate group, such as the terminal phosphate of the oligonucleotide backbone.

[0091] In some specific examples, one of the targeting moiety and the drug molecule is a peptide / protein or an antibody or an antigen-binding fragment thereof, and the other is a double-stranded RNAi molecule. Therefore, in some examples, the targeting moiety described herein is conjugated to the 3' end of the sense strand. In some examples, the targeting moiety described herein is conjugated to the 5' end of the sense strand. In some examples, the targeting moiety described herein is conjugated to the 3' end of the antisense strand. In some examples, the targeting moiety described herein is conjugated to the 5' end of the antisense strand.

[0092] In some instances, the targeting moiety and the drug molecule are peptides / proteins or antibodies or their Fabs. Therefore, in some instances, the targeting moiety and the drug molecule can be expressed as fusion proteins. In some specific instances, the targeting moiety and the drug molecule are directly attached to each other. In some specific instances, the targeting moiety and the drug molecule are connected via insertion of amino acids. In some specific instances, the targeting moiety and the drug molecule are connected by a peptide linker. Specifically, the joint can be connected with the constituent amino acids by its side group (for example, by the disulfide bond with cysteine), and in other instances, when present, the joint is connected with the α-carbon amino and carboxyl groups of the terminal amino acids.

[0093] To produce a fusion protein, this generally involves generating a DNA sequence encoding the fusion protein, placing the DNA in an expression cassette under the control of a specific promoter, expressing the protein in a host, isolating the expressed protein, and, if necessary, renaturing the protein. The DNA encoding the fusion protein can be prepared by any suitable method, including, for example, cloning and restricting appropriate sequences or direct chemical synthesis by methods such as the phosphotriester method described in Narang et al. (1979) Meth. Enzymol. 68: 90-99, the phosphodiester method described in Brown et al. (1979) Meth. Enzymol. 68: 109-151: the diethylphosphoramidite method described in Beaucage et al. (1981) Tetra. Lett., 22: 1859-1862; and the solid support method of U.S. Patent No. 4,458,066. In some instances, DNA amplification methods such as polymerase chain reaction (PCR) can be used to clone DNA encoding the fusion protein constructs described herein. Therefore, for example, the nucleic acid sequence encoding the targeting portion disclosed herein can be PCR amplified using primers containing designed restriction sites. This produces a nucleic acid encoding the targeting portion disclosed herein and having terminal restriction sites. Similarly, a nucleic acid encoding the drug molecule disclosed herein with complementary restriction sites can be provided. The connection and insertion vector of the sequence produces a vector encoding the fusion protein. The nucleic acid sequence encoding the fusion protein can be expressed in a variety of host cells, including E.coli, other bacterial hosts, yeast and various higher eukaryotic cells, such as COS, CHO and HeLa cell lines and myeloma cell lines. The recombinant protein gene will be operably connected to the appropriate expression control sequence of each host. For E. coli, this includes a promoter, such as T7, trp or lambda promoter, a ribosome binding site and preferably a transcription termination signal. For eukaryotic cells, the control sequence will include promoters and preferably enhancers derived from immunoglobulin genes, SV40, cytomegalovirus, etc., as well as polyadenylation sequences, and may include splicing donor and acceptor sequences. Plasmids can be transferred into selected host cells by well-known methods, such as calcium chloride transformation of E. coli and calcium phosphate treatment or electroporation of mammalian cells. Plasmid-transformed cells can be selected by antibiotic resistance conferred by genes (such as amp, gpt, neo and hyg genes) contained on the plasmid.

[0094] Once expressed, the recombinant fusion protein can be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis, etc. (see generally R. Scopes (1982) Protein Purification, Springer-Verlag, NY; Deutscher (1990) Methods in Enzymology Vol. 182: Guide to Protein Purification., Academic Press, Inc. NY). Substantially pure compositions of at least about 90 to 95% homogeneity are preferred, and compositions of 98 to 99% or greater homogeneity are most preferred for pharmaceutical use. Once purified, partially purified, or homogeneous as desired, the polypeptide can be used therapeutically.

[0095] In some instances, certain modifications may be made to the fusion protein without reducing its biological activity. Some modifications may be made to facilitate cloning, expression, or incorporation of a drug molecule or targeting moiety disclosed herein into the fusion protein. Such modifications include, for example, adding a methionine at the amino terminus to provide a start site, or placing additional amino acids at either end to create a restriction site or stop codon for convenient localization.

[0096] In some instances, one of the targeting moiety and the drug molecule is a peptide / protein or an antibody or an antigen-binding fragment thereof, and the other is a small molecule. Exemplary linkers can be found in U.S. Patents 10864279, 9814784, 11173214, 11104968, 9872924, 10537644, U.S. Patent Applications US20180169262, US20220072137, and US20180311375, all of which are incorporated herein by reference.

[0097] In some instances, one of the targeting moiety and the drug molecule is a double-stranded RNAi molecule or a single-stranded antisense oligonucleotide, and the other is a small molecule. Therefore, exemplary linkers are found in U.S. Patents 8772472, 9211343, 8969543, 9074208, 10947323, U.S. Patent Applications US20120177723, US20120108803, US20040053876, US20210108200, and US20150315587, all of which are incorporated herein by reference.

[0098] Other properties of drug conjugates and pharmaceutical compositions

[0099] In some instances, multiple targeting moieties disclosed herein can be attached to a single drug molecule disclosed herein. In some instances, multiple drug molecules disclosed herein can be attached to a single targeting moiety disclosed herein. In some instances, a single targeting moiety disclosed herein is attached to a single drug molecule disclosed herein. In some instances, the ratio of drug molecules to targeting moieties is 1:1. In some instances, the ratio of drug molecules to targeting moieties is about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. In some instances, the ratio of drug molecules to targeting moieties is about 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.

[0100] In some aspects, provided herein are pharmaceutical compositions comprising a drug conjugate disclosed herein and a pharmaceutically acceptable excipient. In some examples, the pharmaceutical composition is formulated for parenteral, intravenous, subcutaneous, intrathecal or intrasciatic injection.

[0101] In some instances, the drug conjugates provided herein are preferably internalized to Schwann cells. In some instances, the drug conjugates provided herein are internalized to Schwann cells at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 times more than the internalization of cell types other than Schwann cells.

[0102] In some instances, the drug conjugates provided herein enter the bloodstream and remain intact. In some instances, the drug conjugates provided herein enter the bloodstream and effectively cross the blood-nerve barrier. In some instances, the drug conjugates provided herein enter the bloodstream, effectively cross the blood-nerve barrier, and are enriched in the sciatic nerve.

[0103] The pharmaceutical compositions described herein can be prepared to include 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, alcohol, glycerol and polyols. Intravenous carriers include liquids and nutritional supplements. Preservatives include antimicrobial agents, antioxidants, chelating agents and inert gases. Other pharmaceutically acceptable vehicles include aqueous solutions, nontoxic excipients, including salts, preservatives, buffers, etc., such as, for example, Remington: The Science and Practice of Pharmacy, 21st Ed., Gennaro, Ed., Lippencott Williams & Wilkins (2005), and The United States Pharmacopeia: The National Formulary (USP 36NF31), published in 2013. The pH and exact concentration of the various components of the pharmaceutical composition are adjusted according to routine techniques in the art. See Goodman and Gilman's, The Pharmacological Basis for Therapeutics.

[0104] The pharmaceutical compositions described herein can be administered topically or systemically. The therapeutically effective amount will vary according to factors such as the degree of infection of the subject, the age, sex, and weight of the individual. The dosage regimen can be adjusted to provide the best therapeutic response. For example, several divided doses may be administered daily, or the dosage may be reduced proportionally, as dictated by the urgency of the treatment situation.

[0105] The pharmaceutical composition can be applied in a convenient manner, such as by injection (e.g., subcutaneous, intravenous, intraorbital, intracapsular, intrathecal, etc.), oral administration, ocular administration, inhalation, topical application, or rectal administration. Depending on the route of administration, the pharmaceutical composition can be coated with materials that protect the pharmaceutical composition from the effects of enzymes, acids, and other natural conditions that may inactivate the pharmaceutical composition. The pharmaceutical composition can also be applied parenterally or intraperitoneally. Dispersions can also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, and in oils. Under common storage and use conditions, these preparations can contain preservatives to prevent the growth of microorganisms.

[0106] Pharmaceutical compositions suitable for injection purposes include sterile aqueous solutions (water soluble) or dispersions and sterile powders for the temporary preparation of sterile injection solutions or dispersions. In some instances, the composition is usually sterile, and fluidity reaches a degree of ease of injection. In some instances, the composition will be stable under manufacturing and storage conditions, and is preserved to resist the contamination of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium, which contains, for example, water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycol, etc.), suitable mixtures thereof and vegetable oils. For example, in the example of a dispersant, the desired particle size is maintained by using a coating such as lecithin, and appropriate fluidity can be maintained by using a surfactant. The effect of microorganisms can be prevented by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many instances, isotonic agents, such as sugars, polyols, 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 which delays absorption, for example, aluminum monostearate and gelatin.

[0107] Sterile injectable solutions can be prepared by mixing the desired amount of the pharmaceutical composition with one or a combination of the ingredients listed above in a suitable solvent as required and then filtering and sterilizing. Typically, dispersions are prepared by mixing the pharmaceutical composition into a sterile carrier that contains a basic dispersion medium and other ingredients listed above.

[0108] In some examples, the pharmaceutical composition is formulated into a dosage unit form for ease of administration and uniform dosage. As used herein, a dosage unit form refers to a physically discrete unit suitable for use as a single dose for a subject to be treated; each unit containing a predetermined amount of a pharmaceutical composition is calculated to produce the desired therapeutic effect in combination with a desired pharmaceutical carrier. The specifications of the dosage unit form are related to the characteristics of the pharmaceutical composition and the specific therapeutic effect to be achieved. An effective amount of the main pharmaceutical composition is mixed with a suitable pharmaceutically acceptable vehicle in an acceptable dosage unit for convenient and effective administration. In the example of a composition containing a supplementary active ingredient, the dosage is determined by reference to the conventional dose and mode of administration of the ingredient.

[0109] The pharmaceutical composition can be administered orally, for example, in a carrier, for example, in an enteric coated unit dosage form. The pharmaceutical composition and other ingredients can also be encapsulated in a hard or soft shell gelatin capsule or compressed into a tablet. For oral therapeutic administration, the pharmaceutical composition can be incorporated with excipients and used in the form of ingestible tablets, lozenges, capsules, pills, wafers, etc. The percentages of the composition and preparation can of course vary, and can conveniently be between about 5% and about 80% of the unit weight. Tablets, lozenges, pills, capsules, etc. can also contain the following: a binder, such as tragacanth, gum arabic, corn starch, or gelatin; an excipient, such as dicalcium phosphate; a disintegrant, such as corn starch, potato starch, alginic acid, etc.; a lubricant, such as magnesium stearate; and a sweetener, such as sucrose, lactose, or saccharin, or a flavoring, such as peppermint, wintergreen oil, or cherry flavoring. When the dosage unit form is a capsule, it can also contain a liquid carrier in addition to the above-mentioned types of materials. Various other materials may exist as coatings or otherwise change the physical form of the dosage unit. For example, tablets, pills or capsules may be coated with shellac, sugar or both. Syrups or elixirs may contain reagents, sucrose as a sweetener, methylparaben and propylparaben as preservatives, dyes and flavorings, such as cherry or orange flavorings. Any material used to prepare any dosage unit form should have pharmaceutically acceptable purity and be substantially nontoxic under the amount employed. In addition, the pharmaceutical composition may be incorporated into a sustained release formulation and a preparation.

[0110] For the polynucleotide molecules 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, and the like; (ii) acid addition salts formed with inorganic acids such as 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, naphthalene disulfonic acid, polygalacturonic acid, and the like.

[0111] In some instances, the drug molecules disclosed herein are encapsulated in a nanoparticle system, and the nanoparticle system includes multiple particles, such as but not limited to polymers, gold particles, silica, lipids or protein-based particles, and multiple particles are formulated to incorporate a targeting portion disclosed herein (see WO 2023 / 056282, the entire contents of which are incorporated herein by reference). In some instances, multiple particles are coupled to a targeting portion disclosed herein by non-covalent binding (e.g., absorption), and this coupling maintains the biological activity of the targeting portion. In some instances, multiple particles are coupled to a targeting portion disclosed herein by covalent binding, and this coupling maintains the biological activity of the targeting portion. In some instances, absorption is a non-covalent fixation strategy, such as but not limited to physical absorption or ion binding. In some instances, physical adsorption includes a connection between a targeting portion disclosed herein and a particle surface by weak interactions (e.g., electrostatic, hydrogen bonding, hydrophobic or van der Waals attraction). In some instances, ion binding is based on an ionic bond between an oppositely charged surface and a particle of a targeting portion disclosed herein. In some instances, covalent binding requires pre-activation of particles. In some instances, covalent binding is based on carbodiimide chemistry, maleimide chemistry (see Jahan ST et al. Int. J. Drug Deliv. 2017, the entire contents of which are incorporated herein by reference), or "click chemistry" (see Yi, G et al. Biomater. Res. 2018, the entire contents of which are incorporated herein by reference). In some specific instances, the targeting moiety comprises an antibody or its antigen-binding fragment as described herein, which is combined with a nanoparticle system using maleimide-thiol chemistry (see Niels Dammes et al. Nat Nanotechnol. 2021, the entire contents of which are incorporated herein by reference). In some specific instances, the targeting moiety disclosed herein is conjugated to the surface of a nanoparticle system comprising multiple polymer nanoparticles (see Nasrul Wathoni, et al. Drug Delivery., 2022, the entire contents of which are incorporated herein by reference).

[0112] In some examples, the drug molecules disclosed herein are encapsulated in extracellular vesicles (EVs), and EVs are modified to present the targeting moieties disclosed herein on their outer surfaces (see WO 2020 / 141369 or Chan., H., et al, Front. Cell. Dev. Biol., 2021, 9, 751079, the entire contents of which are incorporated herein by reference). In some specific examples, the targeting moieties disclosed herein are tethered to the surface of EVs by fusion with lysosomal-associated membrane glycoprotein 2b protein (see Liang., Y et al., ACS Appl Mater Interfaces., 2020, 12, 36938, the entire contents of which are incorporated herein by reference). In some specific examples, the targeting moieties disclosed herein are tethered to the surface of EVs by bioorthogonal chemistry (see Tian., T., et al., Biomaterials, 2018., 150, 137, the entire contents of which are incorporated herein by reference).

[0113] method

[0114] In some aspects, provided herein is a method for targeted delivery of a drug to Schwann cells by contacting a drug conjugate disclosed herein or a pharmaceutical composition comprising a drug conjugate disclosed herein with a Schwann cell. In some instances, when the targeting moiety binds to a cell surface molecule expressed on a Schwann cell, the drug conjugate is internalized into the Schwann cell. In some instances, the cell surface molecule expressed on the Schwann cell is a cell surface receptor with an extracellular domain. In some instances, the cell surface molecule is enriched on the surface of the Schwann cell. In some instances, the drug conjugate is delivered to Schwann cells by systemically delivering a drug conjugate disclosed herein or a pharmaceutical composition disclosed herein to the body of the subject. In some instances, the drug conjugate disclosed herein can penetrate the blood-nerve barrier. In some instances, the drug conjugate disclosed herein can penetrate the blood-nerve barrier and can be enriched in the sciatic nerve. In some aspects, provided herein is a method for preferentially delivering a drug to Schwann cells or to Schwann cells by contacting a drug conjugate disclosed herein or a pharmaceutical composition comprising a drug disclosed herein with a Schwann cell. In some examples, the drug conjugate is preferably internalized into Schwann cells when the targeting moiety is bound to a cell surface molecule expressed on Schwann cells. In some examples, the cell surface molecule preferably expressed on Schwann cells is a cell surface receptor with an extracellular domain. In some examples, the cell surface molecule is preferably expressed on the surface of Schwann cells. In some examples, the cell surface molecule is expressed in Schwann cells at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 2, 3, 4, 5 times more than in other types of cells. In some examples, the method provided herein ensures that the drug is preferably internalized into Schwann cells more than internalized into cell types other than Schwann cells by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 2, 3, 4, 5 times. In some instances, the drug conjugates disclosed herein are delivered to Schwann cells by systemically delivering the drug conjugates disclosed herein or the pharmaceutical compositions disclosed herein to the body of the 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 the blood-nerve barrier and can be enriched in the sciatic nerve.

[0115] In some instances, the targeted delivery methods disclosed herein have improved pharmacokinetics. In specific instances, the targeted delivery methods disclosed herein result in a higher Cmax (i.e., the maximum concentration of the drug molecule in Schwann cells). In specific instances, when Cmax is reached in Schwann cells, the targeted delivery methods disclosed herein result in a shorter Tmax. In specific instances, the targeted delivery methods disclosed herein result in a higher area under the concentration-time curve. In specific instances, the targeted delivery methods disclosed herein result in a longer t1 / 2 (i.e., the time it takes for the concentration of the drug molecule to drop by half once distribution equilibrium is reached in Schwann cells). Thus, in some instances, the targeted delivery methods 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 instances, the targeted delivery methods 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 instances, the targeted delivery methods disclosed herein result in the circulation and availability of the drug conjugate in the subject for up to 90, 120, 150, 180, 210, or 240 days.

[0116] In some instances, the targeted delivery methods disclosed herein result in a reduction in distribution in any other tissue, organ, or cell type other than Schwann cells. In some instances, the targeted delivery methods disclosed herein produce an insignificant amount or minimal amount in any other tissue, organ, or cell type than Schwann cells. In some instances, the targeted delivery methods disclosed herein are non-immunogenic. In some instances, the targeted delivery methods disclosed herein are biodegradable. In some instances, the targeted delivery methods disclosed herein are applicable to a variety of different drug molecules.

[0117] In some examples, the method of targeted delivery is achieved by parenteral, intravenous, subcutaneous, intrathecal or intrasciatic injection of a pharmaceutical composition disclosed herein. In some examples, the method of targeted delivery is achieved by parenteral intravenous injection of a pharmaceutical composition disclosed herein. In some examples, the method of targeted delivery is achieved by local delivery (see, e.g., Pitiot et al. (2022) Antibodies (Basel) 11(3):56.)

[0118] On the other hand, provided herein is a method for treating or preventing a peripheral demyelinating disease or peripheral neuropathy, or alleviating or reducing the symptoms of a peripheral demyelinating disease or peripheral neuropathy in a subject in need thereof, comprising: providing a drug conjugate disclosed herein or a pharmaceutical composition disclosed herein; and administering the drug conjugate or the pharmaceutical composition to the subject to treat the peripheral demyelinating disease or peripheral neuropathy, thereby alleviating or reducing the symptoms of the peripheral demyelinating disease or peripheral neuropathy.

[0119] In some examples, the peripheral demyelinating disease or peripheral neuropathy is CMT disorder (Charcot-Marie-Tooth), Guillain–Barré syndrome (acute inflammatory demyelinating polyradiculopathy type), schwannomatosis, chronic inflammatory demyelinating polyneuropathy, nerve trauma, post-chemotherapy neuropathy, diabetic peripheral neuropathy, migraine, Schwannomas, neurofibromatosis type 1 (NF1), malignant peripheral nerve sheath tumor (MPNST), or nerve injury.

[0120] In some examples where the increase of disease-associated molecules leads to disease, the drug conjugate or pharmaceutical composition is administered at a dose and schedule that is effective to reduce the expression of disease-associated molecules 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 examples where the increase of disease-associated molecules leads to disease, the drug conjugate or pharmaceutical composition is administered at a dose and schedule that is effective to reduce the expression of disease-associated molecules by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%. In some examples where the increase of disease-associated molecules leads to disease, the drug conjugate or pharmaceutical composition is administered at a dose and schedule that is effective to reduce the 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 examples where the increase of disease-associated molecules leads to disease, the drug conjugate or pharmaceutical composition is administered at a dose and schedule that is effective to reduce the 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%.

[0121] In some instances, the drug conjugate or pharmaceutical composition is administered at 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 pharmaceutical composition is administered at 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 pharmaceutical composition is administered at 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 pharmaceutical composition is administered at 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%.

[0122] In some instances, the drug conjugate or pharmaceutical composition is administered at a dose and schedule effective to reduce the expression of anti-ganglioside 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 pharmaceutical composition is administered at a dose and schedule effective to reduce the expression of anti-ganglioside 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 pharmaceutical composition is administered at a dose and schedule effective to reduce the activity of the anti-ganglioside antibody 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 pharmaceutical composition is administered at a dose and schedule effective to reduce the activity of the anti-ganglioside antibody by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%.

[0123] In some examples where the lack of a disease-associated molecule results in a disease, the drug conjugate or pharmaceutical composition is administered at a dose and schedule effective to increase the 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 examples where the lack of a disease-associated molecule results in a disease, the drug conjugate or pharmaceutical composition is administered at a dose and schedule effective to increase the 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 examples where the lack of a disease-associated molecule results in a disease, the drug conjugate or pharmaceutical composition is administered at a dose and schedule effective to increase the 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 examples where the lack of a disease-associated molecule results in a disease, the drug conjugate or pharmaceutical composition is administered at a dose and schedule effective to increase the 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%.

[0124] In some instances, the drug conjugate or pharmaceutical composition is administered at a dose and schedule effective to increase the expression of a subunit of the SWI / SNF protein complex encoded by the 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 pharmaceutical composition is administered at a dose and schedule effective to increase the expression of a subunit of the SWI / SNF protein complex encoded by the 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 pharmaceutical composition is administered at a dose and schedule effective to reduce the activity of a subunit of the SWI / SNF protein complex encoded by the 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 pharmaceutical composition is administered at a dose and schedule effective to reduce the activity of a subunit of the SWI / SNF protein complex encoded by the gene SMARCB1 by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%.

[0125] In some examples, the drug conjugate or pharmaceutical composition is administered at a dose and schedule effective to increase the expression of the LZTR1 protein encoded by the 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 examples, the drug conjugate or pharmaceutical composition is administered at a dose and schedule effective to increase the expression of the LZTR1 protein encoded by the 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 examples, the drug conjugate or pharmaceutical composition is administered at a dose and schedule effective to reduce the activity of the LZTR1 protein encoded by the 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 examples, the drug conjugate or pharmaceutical composition is administered at a dose and schedule effective to reduce the activity of the LZTR1 protein encoded by the gene LZTR1 by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%.

[0126] In some examples, the drug conjugate or pharmaceutical composition is administered at a dose and schedule effective to increase the expression of myelinating marker genes 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 at different stages of development or wound repair and express different Schwann cell-specific markers. Therefore, in a specific example, the drug conjugate or pharmaceutical composition is administered at a dose and schedule effective to increase the expression of marker genes 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 a specific example, the drug conjugate or pharmaceutical composition is administered at a dose and schedule effective to increase the expression of a marker gene of an 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 a specific example, the drug conjugate or pharmaceutical composition is administered at a dose and schedule effective to increase the expression of a marker gene of a 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 certain examples, the drug conjugate or pharmaceutical composition is administered at a dose and schedule effective to increase the expression of marker genes of non-myelinated myelinating Schwann cells 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 examples, the drug conjugate or pharmaceutical composition is administered at a dose and schedule effective to increase the expression of marker genes of non-myelinated myelinating Schwann cells by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%. NTR , Sox10, Sox2, GAP43, NCAM, Krox20, Oct6, MBP, and MPZ) by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% when the drug conjugate or pharmaceutical composition is administered at a dose and schedule that increases the expression of the cytokine receptor antagonist (RACE), Sox10, Sox2, GAP43, NCAM, Krox20, Oct6, MBP, and MPZ) by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 9

[0127] Certain terms

[0128] The term "disease-associated molecule" as used herein generally refers to a molecule that is directly or indirectly related to the pathogenesis, progression, extension or recurrence of a disease. In some instances, the abnormal expression level of a disease-associated molecule is related to the pathogenesis, progression, extension or recurrence of a disease. In some instances, the abnormal activity of a disease-associated molecule is related to the pathogenesis, progression, extension or recurrence of a disease.

[0129] The term "complementary" as used herein generally refers to the ability of accurate pairing between two nucleotides or two groups of nucleotides. In particular, complementarity is a term that characterizes the degree of hydrogen bond pairing that causes the combination between two nucleotides or two groups of nucleotides. For example, if the base at a position of an oligonucleotide can be hydrogen bonded with the base at the corresponding position of a target nucleic acid (such as mRNA), the base at the position is considered to be complementary to each other. Base pairing can include standard Watson-Crick (Watson-Crick) base pairing and non-Watson-Crick base pairing (such as Wobble (Wobble) base pairing and Hoogsteen (Hoogsteen) base pairing). For example, in some instances, for complementary base pairing, adenosine type base (A) is complementary to thymidine type base (T) or uracil type base (U), cytosine type base (C) is complementary to guanosine type base (G), and universal bases such as 3-nitropyrrole or 5-nitroindole can be hybridized with any A, C, U or T and are considered to be complementary. Inosine (I) is also considered in the art to be a universal base and is considered complementary to any of A, C, U, or T.

[0130] As used herein, the term "conservative amino acid substitution" generally refers to an amino acid substitution that does not change the relative charge or size characteristics of the protein to which the amino acid substitution is made. Variants can be prepared according to methods known to those of ordinary skill in the art for altering polypeptide sequences, such as those found in references compiling such methods, e.g., Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Fourth Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2012, or Current Protocols in Molecular Biology, FM Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative substitutions of amino acids include substitutions made in 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.

[0131] The term "antibody" as used herein generally refers to a polypeptide comprising at least one immunoglobulin variable domain or at least one antigenic determinant (e.g., a paratope specifically bound to an antigen). In some instances, the antibody is a full-length antibody. In some instances, the antibody is a chimeric antibody. In some instances, the antibody is a humanized antibody. However, in some instances, the antibody is a Fab fragment, a F(ab')2 fragment, a Fv fragment, or a scFv fragment. In some instances, the antibody is a nanobody derived from a camel antibody or a nanobody derived from a shark antibody. In some instances, the antibody is a double antibody. In some instances, the antibody comprises a framework with a human germline sequence. In other instances, the antibody comprises a heavy chain constant domain selected from the group consisting of: IgG, IgG1, IgG2, IgG2A, IgG2B, IgG2C, IgG3, IgG4, IgA1, IgA2, IgD, IgM, and IgE constant domains. In some examples, the antibody comprises a heavy chain (H) variable region (abbreviated herein as VH) and / or a light chain (L) variable region (abbreviated herein as VL). In some examples, the antibody comprises a constant domain, such as an Fc region. An immunoglobulin constant domain refers to a heavy chain or light chain constant domain. Human IgG heavy chain and light chain constant domain amino acid sequences and functional variants thereof are known. With regard to the heavy chain, in some examples, the heavy chain of the antibody described herein may be an alpha (α), delta (Δ), epsilon (ε), gamma (γ) or mu (μ) heavy chain. In some examples, the heavy chain of the antibody described herein may comprise a human alpha (α), delta (Δ), epsilon (ε), gamma (γ) or mu (μ) heavy chain. In specific examples, the antibody described herein comprises a human γ1 CH1, CH2 and / or CH3 domain. In some examples, the amino acid sequence of the VH domain comprises the amino acid sequence of a human gamma (γ) 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, for example, see U.S. Pat. No. 5,693,780 and Kabat EA et al., (1991) supra. In some examples, the VH domain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or at least 99% identity with any variable chain constant region provided herein. In some examples, the antibody is modified, for example, by glycosylation, phosphorylation, SUMOylation and / or methylation. In some examples, the antibody is a glycosylated antibody bound to one or more sugar or carbohydrate molecules. In some examples, one or more sugar or carbohydrate molecules are conjugated to the antibody via N-glycosylation, O-glycosylation, C-glycosylation, glycosylation (GPI anchor attachment) and / or phosphoglycosylation. In some examples, one or more sugar or carbohydrate molecules are monosaccharides, disaccharides, oligosaccharides or polysaccharides.In some instances, one or more sugars or carbohydrate molecules are branched oligosaccharides or branched polysaccharides. In some instances, one or more sugars or carbohydrate molecules include mannose units, glucose units, N-acetylglucosamine units, N-acetylgalactosamine units, galactose units, fucose units or phospholipid units. In some instances, an antibody is a construct comprising a polypeptide comprising one or more antigen-binding fragments of the present disclosure connected to a linker polypeptide or an immunoglobulin constant domain. The linker polypeptide comprises two or more amino acid residues connected by a peptide bond and is used to connect one or more antigen-binding moieties. Examples of linker polypeptides have been reported (see, e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448; Poljak, RJ, et al. (1994) Structure 2: 1121-1123). In addition, antibody can be a part of a larger immunoadhesion molecule, which is formed by covalent or non-covalent binding of an antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion molecules include the use of streptavidin core regions to prepare tetrameric scFv molecules (Kipriyanov, SM, et al. (1995) Human Antibodies and Hybridomas 6:93-101) and the use of cysteine ​​residues, marker peptides and C-terminal polyhistidine tags to prepare divalent and biotinylated scFv molecules (Kipriyanov, SM, et al. (1994) Mol. Immunol. 31:1047-1058).

[0132] The term "covalent" as used herein generally refers to the characteristic of two or more molecules being linked together by 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 a disulfide bridge) used as a linker between the molecules. However, in some instances, two or more molecules can be covalently linked together by a molecule used as a linker, which links two or more molecules together by multiple covalent bonds. In some instances, the linker can be a cleavable linker. However, in some instances, the linker can be a non-cleavable linker.

[0133] As used herein, the term "specific binding" 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 antibodies, the term "specific binding" refers to the ability of an antibody to bind to a specific antigen with a degree of affinity or avidity compared to an appropriate reference antigen (or antigens), which enables the antibody to be used to distinguish the specific antigen from other antigens, for example, to the extent that allows preferential targeting of Schwann cells by binding to the antigen, as described herein. In some instances, if the K of the antibody binding to the target is D is at least about 10 - 4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13 MM, or lower, the antibody specifically binds to the target.

[0134] As used herein, the terms "(poly)peptide" or "protein" are used interchangeably and generally refer to a series of amino acid residues (i.e., a polymer of amino acids) linked by peptide bonds, and include modified amino acids (e.g., phosphorylated, glycosylated, glycosylated, etc.) and amino acid analogs. Exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, paralogs, fragments and other equivalents, variants and analogs described above.

[0135] As used herein, the terms "subject," "individual," "mammal," and "patient" are used interchangeably and generally refer to and can be humans as well as non-human mammals (e.g., non-human primates, canines, equines, felines, porcines, bovines, ungulates, lagomorphs, etc.). In various examples, a subject can be a person (e.g., an adult male, adult female, adolescent male, adolescent female, boy, girl) under the care of a physician or other health worker in a hospital as an outpatient or other clinical setting. In certain examples, a subject may not be under the care or prescription of a physician or other health worker.

[0136] The term "joint" as used herein generally refers to a molecule used to connect two or more molecules. In some instances, a joint can form a covalent bond with two molecules. Suitable joints include, but are not limited to, straight or branched carbon joints, heterocyclic carbon joints, or peptide joints.

[0137] Whenever the term "at least", "greater than", or "greater than or equal to" appears before the first value in a series of two or more values, the term "at least", "greater than", or "greater than or equal to" applies to each value in the series. 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.

[0138] The terms "a," "an," and "the" include plural references unless the content clearly dictates otherwise.

[0139] In particular the term "about" with respect to a given amount is intended to encompass a deviation of ±10%.

[0140] Although examples of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that these examples are provided only as examples. In examples that do not depart from the present disclosure, those skilled in the art may conceive of many variations, changes and substitutions. It should be understood that various substitutions of the examples of the present disclosure described herein may be adopted.

[0141] Example

[0142] The following is a description of various methods and materials used in the studies, and is presented in order to provide one of ordinary skill in the art with a complete disclosure and description of how to make and use the present disclosure, and is not intended to limit the scope of what the inventors regard as their disclosure, nor is it intended to represent that the following experiments were performed, and are all experiments that could 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.

[0143] Example 1: Evaluation of targeted delivery in vitro and in vivo

[0144] A conjugate of ML-LBP21 and GFP was generated.

[0145] The nucleic acid sequence of ML-LBP21 was synthesized and introduced into a bacterial expression system under the control of a lac promoter with a 6xHis tag at the C-terminus, with and without green fluorescent protein (GFP), and then transformed into BL21 Escherichia coli (E. coli). After induction with IPTG (0.1 mM, 16 h at 26 ° C), the cells were centrifuged and collected. Ultrasonic treatment was used to lyse the cells and collect the soluble fraction. The cell supernatant was further purified on a HisTrap column, then fully washed and eluted with imidazole. The purity of the purified protein was subsequently evaluated by SDS-PAGE and analytical SEC. The binding affinity of ML-LBP21 to the leprosy receptor laminin 2 was also evaluated.

[0146] The delivery ability of the conjugate of ML-LBP21 and GFP to Schwann cells was first tested by incubating the conjugate with primary Schwann cells or cell lines such as the mouse Schwann cell line MSC80 used as a system for neuropathological studies.

[0147] To determine whether ML-LBP21-GFP conjugate or another detectable tag is targeted for delivery to Schwann cells, ML-LBP21-GFP conjugate was co-incubated with primary cells or / and MSC80 cells. As a negative control, cells were incubated with GFP alone. As a positive control, primary or MSC80 cells were incubated with GFP with the help of Lipofectamine reagent (Thermo Fisher). After incubation, the number of GFP-positive cells was detected by flow cytometry.

[0148] The total flux of GFP-positive cells was detected. The number of fluorescence detected in the primary cells or / and MSC80 cell groups incubated with ML-LBP21-GFP conjugate was higher than that of cells incubated with GFP alone, and comparable to that of cells incubated with GFP and lipofectamine reagent. This demonstrates that ML-LBP21 promotes Schwann cell delivery.

[0149] In order to confirm that ML-LBP21 indeed mediates the uptake of ML-LBP21-GFP conjugates into primary cells or / and MSC80 cells, competitive uptake assays were performed. After pre-incubation of primary cells or / and MSC80 cells with a certain concentration range of ML-LBP21 peptides, the cells were treated with ML-LBP21-GFP conjugates, while GFP alone was used as a control. The amount of ML-LBP21-GFP conjugates detected in Schwann cells gradually decreased with the increase in the amount of ML-LBP21 peptide. However, the random low uptake of GFP into cells was not affected by pre-incubation with ML-LBP21 peptides. This result indicates that the ML-LBP21-GFP conjugate enters Schwann cells through a ML-LBP21-mediated process.

[0150] Next, ML-LBP21 and GFP or another detectably labeled conjugate was injected into wild-type mice, and the mice were sacrificed at different time points to detect GFP / Tag signals in different tissues and organs and isolated sciatic nerves. The GFP / Tag signal was more enriched in isolated nerves. Further examination of isolated nerves was performed by co-staining of cell markers that distinguish neurons (axons) and Schwann cells. The GFP / Tag signal was enriched in Schwann cells.

[0151] Similarly, ML-LBP21 was replaced with a targeting moiety that binds to gliomedin, and the above assay was used to test the delivery system targeting gliomedin.

[0152] Example 2: Evaluation of targeted delivery of siRNA molecules in vitro and in vivo

[0153] Conjugates of siRNA targeting a gene (e.g., a housekeeping gene) and a targeting moiety (e.g., a ligand, a carbohydrate ligand, ML-LBP21, an anti-neuromedullary peptide antibody) are produced. Double-stranded RNA (dsRNA) is conjugated to a targeting moiety (e.g., a ligand, a carbohydrate ligand, ML-LBP21, an anti-neuromedullary peptide antibody).

[0154] To determine whether the ML-LBP21-siRNA conjugate was successfully delivered to Schwann cells, the ML-LBP21-siRNA conjugate was contacted with primary Schwann cells or / and MSC80 cells. As a negative control, primary cells or / and MSC80 cells were contacted with naked siRNA molecules. As a positive control, primary and / or MSC80 cells were incubated with GFP with the help of RNAiMax transfection reagent (Thermo Fisher). After incubation, the RNA of the cells was collected and quantified by qPCR. The amount of mRNA of the target gene in cells incubated with ML-LBP21-siRNA conjugate was lower than that in cells incubated with siRNA alone, and was comparable to cells incubated with siRNA using RNAiMax transfection reagent.

[0155] Alternatively, cells were genetically modified to express the target of siRNA tagged with Ruby 3. After incubation, the Ruby 3 signal was reduced when compared to siRNA alone and was comparable to the signal of siRNA with RNAiMax transfection reagent.

[0156] The above experiments demonstrate that ML-LBP21 promotes the delivery of siRNA into Schwann cells.

[0157] Example 3: Evaluation of internalization of targeting moieties in Schwann cells in vitro

[0158] To identify antibodies that bind to and are specifically internalized into Schwann cells, several antibodies that recognize extracellular epitopes of proteins expressed by Schwann cells were tested: mouse anti-myelin-associated glycoprotein (MAG) antibody mAb513 (MAB1567, Merck), rabbit anti-neuromodulin antibody (generated against the olfactory mediator domain of rat neuromodulin), mouse anti-neuromodulin antibody (mAb94, generated against residues 273-287 of rat neuromodulin protein or binding residues 273-287 of rat neuromodulin protein (CVIPNDDTLVGRA)), and mouse anti-nectin-like protein 4 (Necl4, also known as Cadm4) antibody mAb244 / 5 (NeuroMAB). These antibodies were first pre-labeled with cy3-conjugated anti-mouse antibody (red). For rabbit-derived antibodies, Cy3 from Jackson ImmunoResearch Inc. (711-165-152) was used. TM 3AffiniPure donkey anti-rabbit IgG (H+L) antibody; for mouse antibodies, Cy from Jackson ImmunoResearch Inc. (715-165-151) was used TM 3AffiniPure Donkey Anti-Mouse IgG (H+L) Antibody. Live rat Schwann cell cultures were then incubated with these antibodies for 30 minutes, then washed and incubated in growth medium for another 48 hours in an incubator. The cell cultures were then fixed and stained with anti-mouse 488 antibody (green) without cell permeabilization. Thus, the yellow labeling shows the antibody molecules found outside the cells, while the red labeling shows the molecules found inside the cells.

[0159] like Figure 1A and Figure 1B As shown, anti-Necl4 antibody (mAb244 / 5) and anti-neuromedulin antibody (mAb94) can bind to Schwann cells and are effectively internalized into Schwann cells. Figure 1C It was shown that anti-MAG antibody was not internalized into Schwann cells.

[0160] The internalization capacity of anti-neuromedulin antibody (mAb94) was further investigated. Rat Schwann cells were cultured on PLL-coated coverslips and incubated with anti-neuromedulin antibody (mAb94) pre-incubated with cy3-conjugated anti-mouse antibody (red) at 37°C for 30 min. The cells were then washed and incubated in growth medium. Figure 1DThe cells were then fixed. The fixed cells were labeled with anti-mouse 488 antibody in instances where 0.1% Triton X100 was present or absent in the antibody solution. Triton is a detergent that permeabilizes the cell membrane, so the cell membrane remains intact in instances without Triton, and only anti-neuromodulin antibody (mAb94) molecules found outside the cell will be co-labeled with the 488 antibody (green) and the red label, while those Ab molecules that have been internalized will only be labeled in red. In other words, experiments without cell permeabilization distinguish between extracellular and intracellular molecules, while experiments with cell permeabilization label the entire antibody population. As Figure 1D As shown, while approximately half of the molecules were still co-labeled with 488 without permeabilization at 24 hours, half of the molecules were found outside the cells. At 72 hours, a significant portion of the antibody population was internalized and therefore not co-labeled with 488 in instances without cell permeabilization. In other words, co-localization of green and red signals indicates internalization of the antibody into the cell only in the presence of Triton X and not in the absence of Triton X (e.g., 72 h).

[0161] Next, the subcellular localization of internalized anti-neuromedulin antibody (mAb94) was investigated at 72 h. Figure 1E As shown, partial colocalization of anti-neuromedulin antibody (mAb94) with the lysosomal marker Lamp1 indicated that anti-neuromedulin antibody (mAb94) entered the lysosome.

[0162] In addition, to test the internalization of the targeting moiety without pre-incubation with the labeled secondary antibody, Cyanine5 NHS was covalently conjugated and labeled with anti-neuromodulin antibody (mAb94) and control mouse IgG antibody. Antibody samples in PBS were mixed with 10X NaHCO3 solution (1M pH; 8.3) and Cyanine5 NHS (Lumiprobe catalog number 43020), which was prepared as a stock solution (2.66 mg / ml DMSO) at a molar ratio of fluorescent dye to antibody of 7:1. The tube with the reaction mixture was kept in the dark and incubated at room temperature for 1 h with continuous mixing. The labeling reaction was terminated after 1 hour by adding 5 ml 1M Tris-HCl pH 7.1 and dialyzed with PBS buffer.

[0163] Rat Schwann cells were incubated with covalently labeled anti-neuromedulin antibody (mAb94) and control IgG antibody (red) at 37°C for 30 minutes and then incubated in growth medium for 24 hours. The cells were fixed and stained with anti-mouse 488 without cell permeabilization to label extracellular antibody molecules. Figure 1FAs shown, the Cy3-labeled mAb94 antibody did not co-localize with the 488 signal, indicating that it was internalized into Schwann cells.

[0164] Example 4: Evaluation of the specificity of anti-neuromedullary peptide antibody (mAb94)

[0165] An incubation setup similar to that disclosed in Example 3 was applied to primary cultures containing sensory neurons and Schwann cells, the cells being generated from dorsal root ganglia (DRG) isolated from wild-type ("WT") or neuromedin null mouse embryos ("KO"). Anti-neurmedin antibody (mAb94) was added to the cell cultures and the cultures were further incubated. In instances where membrane permeabilization was not performed, anti-mouse 488 antibody (green) was applied, followed by a second fixation and labeling with Schwann cell marker antibody ("MAG") or neuronal marker Ab ("neurofilament"). As Figure 2A and Figure 2B As shown, anti-neuromedulin antibody (mAb94) was specifically internalized into Schwann cells in mixed DRG neuron preparations. No signal from anti-neuromedulin antibody (mAb94) was detected in receptor-free DRG cultures.

[0166] Cell type specificity was further confirmed. DRG myelinating cultures isolated from E13 mouse embryos contain 3 cell types: Schwann cells, sensory DRG neurons, and fibroblasts. Anti-neuromodulin antibody (mAb94) antibody (green) was applied to live DRG cultures for 30 minutes at 37°C. The cultures were then fixed and stained with marker antibodies (red) to distinguish neurons (neurofilament "NFH"), fibroblasts (vimentin "Vim"), and Schwann cells ("Cadm4"). The signal from the mAb94 antibody colocalized only with the Schwann cell marker antibody and was not detected at all in neuromodulin null cultures, indicating that the antibody only recognizes neuromodulin and does not cross-react with other proteins (see Figure 2C ).

[0167] In addition, anti-neuromodulin antibody (mAb94) recognizes neuromodulin from different species. COS7 cells were transfected with rat or human neuromodulin. The cells were allowed to bind the extracellular domain of NF186 (NF186FC, red). The cells were then washed, fixed and labeled with anti-neuromodulin antibody (mAb94). The mAb94 antibody binds rat and human neuromodulin.

[0168] Example 5: In vivo evaluation of internalization of anti-neuromedulin antibody (mAb94) into Schwann cells

[0169] To test the targeting of anti-neuromedullary peptide antibody (mAb94) to Schwann cells in vivo, 150 μg of 650-mAb94 antibody (mice #1-3 and #4-6), control 650-IgG (#7-9 and #10-12) or none (mice #13-15) were injected via the tail vein. Mice were killed 24 or 48 hours after injection. Sciatic nerves were collected, fixed and spread (brushed) at single fiber imaging resolution. Treated samples were labeled with anti-caspr antibody (green) that marks the nodes of Ranvier of myelinated nerve fibers. In the nerves of mice injected with 650mAb94, anti-neuromedullary peptide antibody (mAb94) (red) was strongly detected at the nodes of Ranvier and clusters along the entire myelin unit (see Figure 4A , mice #1-3, 24 hours, Figure 4B , mice #4-6, 48 hours). The data indicate that mAb94 antibody penetrates nerves and binds to neuromodulatory peptides on Schwann cells. In contrast, in control injected mice (see Figure 4A , mice #7-9, 24 hours, Figure 4B , mice #10-12, 48 hours) or mice not injected (see e.g. Figure 4C No significant signal was detected in the nerves of mouse #13 in the 1999 study. Clearly, only the anti-neuromedulin antibody (mAb94) showed binding to myelinated nerves at the nodes of Ranvier and along the Schwann cell internodes. However, the control IgG antibody did not show any specific signal and had minimal staining similar to that of uninjected mice.

[0170] Therefore, these data suggest that the anti-neuromedulin antibody (mAb94) is able to cross the blood-nerve barrier, reach the nerves, and be internalized into Schwann cells.

[0171] In addition, the distribution of anti-neuromedulin antibody (mAb94) in different organs was evaluated using fluorescence microscopy.

[0172] Samples of different organs from 15 mice (n=75) were harvested after fluorescent labeling, fixed in 4% paraformaldehyde (PFA) and kept in fixative for 48 hours for further fixation. The tissues were trimmed, placed in embedding cassettes, and processed for paraffin embedding according to the protocol shown below. Four cassettes were prepared for each animal.

[0173] To preserve the fluorescent signal, the tissue processor was modified to a very short protocol: 1. Formalin at 37°C for 10 min; 2. Water treatment for 2 min; 3. 70% ethanol at 45°C for 5 min; 4. 80% ethanol at 45°C for 5 min; 5. 95% ethanol at 45°C for 5 min; 6. 100% ethanol at 45°C for 5 min; 7. 100% ethanol at 45°C for 5 min; 8. 100% ethanol at 45°C for 5 min; 9. Xylene at 45°C for 3 min; 10. Xylene at 45°C for 3 min; 11. Xylene at 45°C for 5 min; 12. Paraffin at 65°C for 5 min; 13. Paraffin at 65°C for 5 min; and 14. Paraffin at 65°C for 10 min.

[0174] Paraffin sections (4 micrometers thick) were cut and placed on glass slides. Fluorescence microscopy was used to evaluate the sections for the presence of fluorescently labeled anti-neuromedulin antibody (mAb94). Figure 4D The photographs shown in Figure 4F were taken using an Olympus microscope (BX60, serial number 7D04032) equipped with a microscope camera (Olympus DP73, serial number OH05504) at objective magnifications of X10 and X20. The number of fluorescently labeled cells at X40 objective magnification was scored using a semi-quantitative system: Grade 0: no positively labeled cells were detected; Grade 1: 1-30 labeled cells were detected; Grade 2: 31-100 labeled cells were detected; Grade 3: 101-150 labeled cells were detected; Grade 4: >150 labeled cells were detected. Table 1 summarizes the specific localization and distribution of the detection antibodies.

[0175] Table 1: Evaluation of anti-neuromedulin antibody (mAb94) labeled cells for each mouse

[0176]

[0177]

[0178]

[0179]

[0180] In general, clear Cy5 signals were found mainly in some organs of groups 1 and 2. In peripheral nerves, clear signals were found in nerve fibers, which were diffuse along the fibers. In the liver, strong signals were found only in the blood sinusoids, but the reaction was also found in negative controls and naive animals. In the spleen, signals were also found in the red pulp, but more detailed examination showed that the red blood cells emit autofluorescence. In the kidney and pancreas, no signals were found at all.

[0181] The signals of neuro (anti-neuromedulin antibody (mAb94)) in group 1 were mostly positive (5 / 6), with stronger signals recorded at 48 hours post injection compared to 24 hours post injection. In group 2 (isotype control), one animal showed very weak signal, probably because the blocking process of the isotype Ab was not complete in the animal. In group 3 (naive), animals were found to be negative.

[0182] In total, fluorescently labeled anti-neuromedulin antibody (mAb94) was injected intravenously in 12 animals. Significant and reliable signals were observed only in peripheral nerves. The signal at 48 hours after injection was stronger than that at 24 hours.

[0183] Example 6: Evaluation of internalization of anti-laminin α-2 antibodies into Schwann cells

[0184] The ability of antibodies binding to laminin α-2 to bind to and be internalized by Schwann cells was further investigated.

[0185] The candidate antibody, without excluding other possible candidate antibodies, can be the antibody ABIN7439129 targeting amino acids 2901-3106 of laminin alpha-2, an antibody against laminin-alpha 2G5 (2D4) from Abnova (Taipei, Taiwan, China), or a laminin alpha-2 monoclonal antibody (Thermo Fisher, #CL3450).

[0186] To evaluate the potential of the tested anti-laminin alpha-2 antibodies (e.g., Invitrogen CL3450) to bind to Schwann cells, rat sciatic nerve sections were used. Sciatic nerves were excised and fixed in 4% PFA for 20 minutes at room temperature (RT). The nerves were washed with PBS and spread (brushed) on a slide to achieve single fiber isolation. The brushed preparations were dried for 2 hours and stored at -20°C until use. For immunofluorescence labeling, the slides were incubated in blocking solution (PBS, 5% normal goat serum, 0.5% TritonX100) at room temperature for 1 hour. The slides were then washed with PBS and incubated with the primary antibody: the tested anti-laminin alpha-2 antibodies and anti-caspr antibodies were labeled at 4°C overnight to mark myelinated axons at the nodes of Ranvier. The primary antibody was diluted in an antibody solution (PBS, 5% normal goat serum, 0.1% Tritonx100). The slides were then washed with PBS and incubated with a secondary antibody diluted in an antibody solution for 45 minutes at room temperature. The slides are then washed with PBS and mounted with, for example, fluoromount-G (Invitrogen).The signal from the tested anti-laminin alpha-2 antibodies is analyzed in relation to structures such as the nodes of Ranvier and the basal lamina covering myelinating Schwann cells.

[0187] The in vitro internalization ability of the tested anti-laminin alpha-2 antibodies was also studied. Neuronal cells (including Schwann cells) were freshly dissected from the sciatic nerve of mice, cultured on PLL-coated coverslips, and incubated with pre-labeled anti-laminin alpha-2 antibodies. The neuronal cells were then washed and incubated in growth medium for different amounts of time (e.g., 24h, 48h, or 72h). The neuronal cells were then fixed and labeled with different permeabilization conditions with a secondary antibody that recognizes the tested anti-laminin-2 antibodies in the presence or absence of 0.1% Triton X-100. Under different incubation times without permeabilization, the percentage of the tested anti-laminin alpha-2 antibodies that were not co-labeled with the secondary antibody was compared.

[0188] In addition, the cell type specificity of the tested anti-laminin-2 antibodies was tested. Similar incubation settings were applied to primary cultures containing sensory neurons, fibroblasts and Schwann cells, which may be generated by DRG isolated from wild-type or laminin-2 null mouse embryos. Cell type markers, such as neurons (neurofilaments), fibroblasts (vimentin) and Schwann cells (MAGs), are used to distinguish different cell types.

[0189] In addition, the in vivo internalization capacity of the tested anti-laminin alpha-2 antibodies was also studied. Mice were grouped and injected with labeled anti-laminin alpha-2 antibodies, and the control was labeled IgG or no injection. Mice were killed 24 or 48 hours after injection. Sciatic nerves were collected, fixed and unfolded (brushed) at single fiber imaging resolution. The treated samples were labeled with anti-caspr antibodies that mark the nodes of Ranvier with myelinated nerve fibers. The signal from the labeled anti-laminin-2 antibody was detected.

[0190] Example 7: Evaluation of internalization of anti-Cadm4 / Necl4 antibodies into Schwann cells

[0191] The internalization ability of the antibody binding to Cadm4 / Necl4 in Schwann cells was further investigated. Figure 1AThe antibody mAb244 / 5 (NeuroMAB) that showed promising internalization results in the present invention is used. Other candidate antibodies, without excluding other possible antibodies, can be Necl4-Fc antibodies from Eshed et al., Neuron, volume 47, issue 2, p215-229, anti-SynCAM4 antibodies (Biolegand, #833302), or IGSF4C / SynCAM4 antibodies (rndsystems, #MAB41642). Schwann cells were cultured on PLL-coated coverslips and incubated with pre-labeled anti-Cadm4 / Necl4 antibodies. The cells were then washed and incubated in growth medium for different amounts of time (e.g., 24h, 48h, or 72h). The cells were then fixed and labeled with different permeabilization conditions in the presence or absence of 0.1% Triton X-100 with a secondary antibody that recognizes the tested anti-Cadm4 / Necl4 antibodies. The percentage of tested anti-Cadm4 / Necl4 antibodies that were not co-labeled with the secondary antibody was compared at different incubation durations without permeabilization.

[0192] In addition, the cell type specificity of the tested anti-Cadm4 / Necl4 antibodies was tested. Similar incubation settings were applied to primary cultures containing sensory neurons, fibroblasts and Schwann cells, which could be generated from DRG isolated from wild-type or Cadm4 / Necl4 null mouse embryos. Cell type markers, such as neurons (neurofilaments), fibroblasts (vimentin) and Schwann cells (MAGs), were used to distinguish different cell types.

[0193] In addition, the in vivo internalization ability of the tested anti-Cadm4 / Necl4 antibodies was further studied. The mice were grouped and injected with labeled anti-Cadm4 / Necl4 antibodies, and the control was labeled IgG or no injection. The mice were killed 24 or 48 hours after injection. The sciatic nerves were collected, fixed and unfolded (brushed) at single fiber imaging resolution. The treated samples were labeled with anti-caspr antibodies that marked the nodes of Ranvier with myelinated nerve fibers. The signal from the labeled anti-Cadm4 / Necl4 antibody was detected.

[0194] It should be understood that the different aspects of the present disclosure can be understood individually, collectively or in combination with each other. The various aspects of the present disclosure described herein can be applied to any specific application disclosed herein. The material compositions disclosed herein in the composition part of the present disclosure can be used for the method part, including the use and production methods disclosed herein, or vice versa.

[0195] Although preferred embodiments of the present disclosure have been shown and described herein, it is obvious to those skilled in the art that these embodiments are provided as examples only. The present disclosure is not intended to be limited by the specific embodiments provided in the specification. Although the present disclosure has been described with reference to the above description, the description and explanation of the embodiments herein are not meant to be limiting. In the embodiments without departing from the present disclosure, those skilled in the art will now think of many changes, modifications and substitutions. In addition, it should be understood that all aspects of the present disclosure are not limited to the specific description, configuration or relative proportion described herein, which depends on various conditions and variables. It should be understood that various alternatives of the embodiments of the present disclosure described herein can be adopted when practicing the present disclosure. Therefore, it is expected that the present disclosure will also cover any such substitutions, modifications, changes or equivalents. It is hoped that the following claims will define the scope of the present disclosure, and therefore cover methods and structures and their equivalents within the scope of these claims.

Claims

1. A drug conjugate comprising a targeting moiety conjugated to a drug molecule, wherein the targeting moiety binds to a receptor expressed on Schwann cells to mediate targeted delivery of the drug conjugate to Schwann cells, and wherein the drug molecule modifies the expression or activity of a disease-related molecule in Schwann cells.

2. The drug conjugate according to claim 1, wherein The receptor is selected from the group consisting of leprosy receptors (eg laminin 2, P0 protein), neuromodulatory peptides (gliomedin) or cell adhesion molecules (Cadm).

3. The drug conjugate according to claim 2, wherein The receptors are neuromodulatory peptides.

4. The drug conjugate according to claim 2, wherein The receptor is a cell adhesion molecule.

5. The drug conjugate according to claim 2, wherein The receptor is a leprosy receptor.

6. The drug conjugate according to any one of claims 1 to 5, wherein The targeting moiety is an antibody or an antigen binding fragment thereof or a ligand molecule.

7. The drug conjugate according to claim 6, wherein The antibody or antigen-binding fragment thereof comprises a monovalent Fab', a bivalent Fab2, a single chain variable fragment (scFv), a diabody, a minibody, a nanobody, a single domain antibody (sdAb) or a camelid antibody or a binding fragment or an antibody mimetic thereof.

8. The drug conjugate according to any one of claims 1 to 6, wherein The targeting moiety is a ligand molecule, and wherein the ligand molecule comprises a peptide, a glycoprotein, a carbohydrate moiety, or a synthetic small molecule.

9. The drug conjugate according to any one of claims 1 to 8, wherein The drug molecule comprises a peptide or a small molecule.

10. The drug conjugate according to any one of claims 1 to 8, wherein The drug molecule comprises a double-stranded RNAi molecule or a single-stranded antisense oligonucleotide.

11. The drug conjugate according to any one of claims 1 to 10, wherein The drug molecule is conjugated to the targeting moiety via a linker.

12. The drug conjugate according to claim 11, wherein The linker is a cleavable linker or a non-cleavable linker.

13. The drug conjugate according to any one of claims 1 to 12, wherein: The ratio of the drug molecule to the targeting moiety is 1:1, 2:1 or 1:

2.

14. A pharmaceutical composition comprising: the drug conjugate according to any one of claims 1 to 13, and a pharmaceutically acceptable excipient.

15. The pharmaceutical composition according to claim 14, wherein The pharmaceutical composition is formulated for parenteral, intravenous, subcutaneous, intrathecal or intrasciatic injection.

16. A method for targeted delivery of a drug to Schwann cells, comprising contacting the drug conjugate according to any one of claims 1 to 13 or the drug composition according to any one of claims 14 to 15 with Schwann cells, wherein the drug molecule comprises the drug.

17. The method according to claim 16, characterized in that The drug is internalized into the Schwann cells through the binding of the targeting moiety to the receptor on the Schwann cells.

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