Methods for Targeted Delivery to Schwann Cells and Treatment of Schwann Cell-Related Diseases

Abstract

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 alters the expression or activity of a disease-related molecule in Schwann cells, is disclosed herein. Further, a method for targeted delivery of a drug to Schwann cells is disclosed herein.

Description

Technical Field

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 388,543, filed Jul. 12, 2022, which is incorporated herein by reference in its entirety.

Background Art

[0002] Schwann cells, also known as neurilemma cells, form the myelin sheath around the axons of the peripheral nervous system. They play important roles in the pathology of various inflammatory, metabolic, or genetic neuropathies (see, e.g., Lehmann and Hoke, CNS Neurol Disord Drug Targets, December 2010, 9(6):801-806; Kamil K et al., Front. Neurol. 2019, 10:87, which are incorporated herein by reference in their entireties).

[0003] Some peripheral neuropathies are associated with abnormal protein or gene expression in Schwann cells, and suppression or regulation of such protein or gene expression in Schwann cells has been considered for treating 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 concerns.

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0005] Therefore, there is a need to specifically target Schwann cells and manage, treat or alleviate a wide range of Schwann cell-related diseases.

Means for Solving the Problems

[0006] (Summary of the Disclosure) Recognizing the need for more effective and more targeted delivery of therapeutic compositions to Schwann cells, the present disclosure, in one aspect, 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 the drug molecule modifies the expression or activity of disease-related molecules in Schwann cells.

[0007] In some cases, the receptor is selected from the group consisting of the leprosy receptor (e.g., laminin 2, P0 protein), gliomedin or a cell adhesion molecule (Cadm). In some cases, the receptor is gliomedin. In some cases, the receptor is Cadm. In some cases, the receptor is the leprosy receptor.

[0008] In some cases, the targeting moiety is an antibody or an antigen-binding fragment thereof, or a ligand molecule. In some cases, the antibody or an antigen-binding fragment thereof includes a monovalent Fab’, bivalent Fab2, single-chain variable fragment (scFv), diabody, minibody, nanobody, single domain antibody (sdAb) or camelid antibody or a binding fragment thereof or an antibody mimetic thereof. In some cases, the targeting moiety is a ligand molecule, where the ligand molecule includes a peptide, glycoprotein, carbohydrate moiety, dendrimer or synthetic small molecule.

[0009] In some cases, the drug molecule includes a peptide or a small molecule. In some cases, the drug molecule includes a double-stranded RNAi molecule or a single-stranded antisense oligonucleotide.

[0010] In some cases, the drug molecule is conjugated to the targeting moiety via a linker. In some cases, the linker is a cleavable linker or a non-cleavable linker. In some cases, the ratio of the drug molecule to the targeting moiety is 1:1, 2:1 or 1:2.

[0011] In some aspects, provided herein is a pharmaceutical composition comprising a drug conjugate described herein and a pharmaceutically acceptable excipient. In some cases, the pharmaceutical composition is formulated for parenteral, intravenous, subcutaneous, intrathecal or intraischial injection.

[0012] In some aspects, provided herein is a method for targeted delivery of a drug to Schwann cells, comprising contacting a Schwann cell with a drug conjugate described herein or a pharmaceutical composition described herein, wherein the drug molecule comprises a drug or a drug precursor. In some cases, when the targeting moiety binds to a receptor on the Schwann cell, the drug or the drug precursor is internalized into the Schwann cell and exerts its biological activity.

[0013] Incorporation by reference All publications, patents and patent applications mentioned herein are hereby incorporated by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference. To the extent that any incorporated publication and patent or patent application contradicts the disclosure contained herein, this specification is intended to supersede and / or prevail over any such conflicting material.

[0014] This application includes at least one drawing created in color. Copies of this patent or patent application publication with one or more 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 set forth in detail in the appended claims. A more thorough understanding of the features and advantages of the present disclosure can be obtained by reference to the following detailed description which illustrates exemplary embodiments in which the principles of the present disclosure are utilized and the accompanying drawings (also referred to herein as "Figure" and "Fig.").

Brief Description of the Drawings

[0016]

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Mode for Carrying Out the Invention

[0017] Detailed Description As used herein, there is provided 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 the 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. Further provided herein is a method for targeted delivery of a drug to Schwann cells. Also provided herein is a method for delivering a drug to Schwann cells by conjugating the drug to a targeting moiety that specifically or preferentially targets Schwann cells. Also provided herein is a method for treating or preventing a 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.

[0018] Conjugate Targeting moiety Historically, for example, the treatment of peripheral nervous system (PNS) disorders associated with axonal loss, axonal dysfunction, or abnormal axon-Schwann cell interactions has focused on targeting axons, but the treatment has been shown to have poor clinical outcomes. In certain PNS disorders where axonal dysfunction is associated with Schwann cell loss, or PNS disorders directly or indirectly associated with Schwann cell dysfunction, the effectiveness and outcome of treatment can be improved by specific or preferential targeting of drugs to Schwann cells.

[0019] However, the specificity of Schwann cell manipulation and / or modification has been the greatest obstacle in advancing Schwann cell therapy. To meet such a need, in some cases, targeting moieties that specifically direct cargo to Schwann cells are provided herein.

[0020] In some embodiments, the targeting moieties disclosed herein bind to receptors expressed on Schwann cells. In some embodiments, the targeting moieties disclosed herein bind to a part of a receptor complex expressed on Schwann cells. In some cases, the receptor is a transmembrane molecule that includes an extracellular domain, and the targeting moiety binds to the extracellular domain of the receptor. In some cases, the receptor complex includes 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 cases, the targeting moiety binds to the extracellular molecule of the complex. In some cases, the targeting moiety binds to a cell adhesion molecule that mediates the interaction between myelinating Schwann cells and the axons they ensheathe. In some cases, the receptor is expressed in myelinating Schwann cells. In some cases, the receptor is highly expressed at the ends of myelinating cells. In some cases, the receptor mediates axon-glia interactions or axon-glia cell contacts. In some cases, the receptor mediates the uptake of a pathogen (or a part thereof) into Schwann cells in a particular pathological condition. Thus, as used herein, a targeting moiety that binds to a receptor also includes a targeting moiety that binds to a part of a receptor complex that naturally exists in the healthy or diseased state of Schwann cells.

[0021] In some cases, the targeted moiety disclosed herein binds to the leprosy receptor. In certain cases, the targeted moiety disclosed herein binds to laminin 2. In certain cases, the targeted moiety disclosed herein binds to the C-terminal region of laminin 2. In some cases, the targeted moiety disclosed herein binds to the α2 subunit of laminin 2. In some cases, the targeted moiety disclosed herein binds to the G domain of the α2 subunit of laminin 2. In some cases, the targeted moiety disclosed herein binds to the G1 domain of the α2 subunit of laminin 2. In some cases, the targeted moiety disclosed herein binds to the G2 domain of the α2 subunit of laminin 2. In some cases, the targeted moiety disclosed herein binds to the G3 domain of the α2 subunit of laminin 2. In some cases, the targeted moiety disclosed herein binds to the G4 domain of the α2 subunit of laminin 2. In some cases, the targeted moiety disclosed herein binds to the G5 domain of the α2 subunit of laminin 2. In some cases, the targeted moiety disclosed herein binds to the β1 subunit of laminin 2. In some cases, the targeted moiety disclosed herein binds to the γ1 subunit of laminin 2. In some cases, the targeted moiety disclosed herein binds to amino acids 2901-3106 of laminin 2. In other cases, the targeted moiety disclosed herein binds to the P0 protein (myelin protein zero). In some cases, the targeted moiety disclosed herein binds to the N-terminal extracellular immunoglobulin (Ig)-like domain of the P0 protein. In some cases, the targeted moiety disclosed herein binds to N-linked oligosaccharides within the extracellular domain of the leprosy receptor or any of its subunits, and / or to the addition of sulfate groups, acyl groups, and phosphate groups.

[0022] In some cases, the targeting moiety disclosed herein is an antibody that binds to the leprosy receptor or an antigen-binding fragment thereof. In certain cases, the targeting moiety disclosed herein is an antibody that binds to laminin 2 or an antigen-binding fragment thereof. In certain cases, the targeting moiety disclosed herein is an antibody that binds to the C-terminal region of laminin 2 or an antigen-binding fragment thereof. In some cases, the targeting moiety disclosed herein is an antibody that binds to the α2 subunit of laminin 2 or an antigen-binding fragment thereof. In some cases, the targeting moiety disclosed herein is an antibody that binds to the G domain of the α2 subunit of laminin 2 or an antigen-binding fragment thereof. In some cases, the targeting moiety disclosed herein is an antibody that binds to the G1 domain of the α2 subunit of laminin 2 or an antigen-binding fragment thereof. In some cases, the targeting moiety disclosed herein is an antibody that binds to the G2 domain of the α2 subunit of laminin 2 or an antigen-binding fragment thereof. In some cases, the targeting moiety disclosed herein is an antibody that binds to the G3 domain of the α2 subunit of laminin 2 or an antigen-binding fragment thereof. In some cases, the targeting moiety disclosed herein is an antibody that binds to the G4 domain of the α2 subunit of laminin 2 or an antigen-binding fragment thereof. In some cases, the targeting moiety disclosed herein is an antibody that binds to the G5 domain of the α2 subunit of laminin 2 or an antigen-binding fragment thereof. In some cases, the targeting moiety disclosed herein is an antibody that binds to the β1 subunit of laminin 2 or an antigen-binding fragment thereof. In some cases, the targeting moiety disclosed herein is an antibody that binds to the γ1 subunit of laminin 2 or an antigen-binding fragment thereof. In some cases, the targeting moiety disclosed herein is an antibody that binds to amino acids 2901-3106 of laminin-2 or an antigen-binding fragment thereof. In other cases, the targeting moiety disclosed herein is an antibody that binds to the P0 protein (myelin protein zero) or an antigen-binding fragment thereof.In some cases, the targeting moiety disclosed herein is an antibody or an antigen-binding fragment thereof that binds to the N-terminal extracellular immunoglobulin (Ig)-like domain of the P0 protein. In some cases, the targeting moiety disclosed herein is an antibody or an antigen-binding fragment thereof that binds to an N-linked oligosaccharide within the extracellular domain of the leprosy receptor or any of its subunits, and / or to the addition of sulfate, acyl, and phosphate groups.

[0023] In some cases, the targeting moiety disclosed herein is a commercially available antibody that binds to laminin-2. In some cases, the targeting moiety disclosed herein is the anti-laminin antibody ABIN7439129. In some cases, the targeting moiety disclosed herein is an anti-laminin alpha 2 monoclonal antibody (Thermo Fisher, #CL3450).

[0024] In some cases, the targeting moiety disclosed herein binds to gliomedin. In some cases, the targeting moiety disclosed herein binds to the extracellular region of gliomedin. In some cases, the targeting moiety disclosed herein binds to the C-terminal region of gliomedin. In some cases, the targeting moiety disclosed herein binds to a portion of the C-terminal region of gliomedin. In some cases, the targeting moiety disclosed herein binds to the coiled-coil domain of gliomedin. In some cases, the targeting moiety disclosed herein binds to the interrupted collagen repeat domain of gliomedin. In some cases, the targeting moiety disclosed herein binds to the olfactomedin domain of gliomedin. In some cases, the targeting moiety disclosed herein binds to amino acid residues 273-287 of rat gliomedin NP_852047.2 (CVIPNDDTLVGRA). In some cases, the targeting moiety disclosed herein binds to amino acid residues 365-460 of gliomedin.

[0025] In some cases, the targeting moiety disclosed herein is an antibody that binds to gliomedin or an antigen-binding fragment thereof. In some cases, the targeting moiety disclosed herein is an antibody that binds to the extracellular region of gliomedin or an antigen-binding fragment thereof. In some cases, the targeting moiety disclosed herein is an antibody that binds to the C-terminal region of gliomedin or an antigen-binding fragment thereof. In some cases, the targeting moiety disclosed herein is an antibody that binds to a portion of the C-terminal region of gliomedin or an antigen-binding fragment thereof. In some cases, the targeting moiety disclosed herein is an antibody that binds to the coiled-coil domain of gliomedin or an antigen-binding fragment thereof. In some cases, the targeting moiety disclosed herein is an antibody that binds to the interrupted collagen repeat domain of gliomedin or an antigen-binding fragment thereof. In some cases, the targeting moiety disclosed herein is an antibody that binds to the olfactomedin domain of gliomedin or an antigen-binding fragment thereof. In some cases, the targeting moiety disclosed herein is an antibody that binds to amino acid residues 273-287 of rat gliomedin NP_852047.2 (CVIPNDDTLVGRA) or an antigen-binding fragment thereof. In some cases, the targeting moiety disclosed herein is an antibody that binds to amino acid residues 365-460 of gliomedin or an antigen-binding fragment thereof.

[0026] In some cases, the targeting moiety disclosed herein is a commercially available antibody that binds to gliomedin. In some cases, the targeting moiety disclosed herein is the anti-gliomedin antibody (mAb94) disclosed in Eshed et al., Neuron, Vol. 47, No. 2, pp. 215-229, which is incorporated herein by reference in its entirety. In some cases, the targeting moiety disclosed herein is NovoPro #175453 of the anti-GLDN antibody. In some cases, the targeting moiety disclosed herein is Thermo Fisher #BS-11032R of the anti-GLDN antibody.

[0027] In some cases, the targeting moieties disclosed herein bind to cell adhesion molecule (Cadm). In some cases, the targeting moieties disclosed herein bind to Cadm1. In some cases, the targeting moieties disclosed herein bind to the IgV set domain of Cadm1 or a portion thereof. In some cases, the targeting moieties disclosed herein bind to the IgC1 set domain of Cadm1 or a portion thereof. In some cases, the targeting moieties disclosed herein bind to the IgI set domain of Cadm1 or a portion thereof. In certain cases, the targeting moieties disclosed herein bind to Cadm2. In some cases, the targeting moieties disclosed herein bind to the IgV set domain of Cadm2 or a portion thereof. In some cases, the targeting moieties disclosed herein bind to the IgC1 set domain of Cadm2 or a portion thereof. In some cases, the targeting moieties disclosed herein bind to the IgI set domain of Cadm2 or a portion thereof. In some cases, the targeting moieties disclosed herein bind to Cadm3. In some cases, the targeting moieties disclosed herein bind to the IgV set domain of Cadm3 or a portion thereof. In some cases, the targeting moieties disclosed herein bind to the IgC1 set domain of Cadm3 or a portion thereof. In some cases, the targeting moieties disclosed herein bind to the IgI set domain of Cadm3 or a portion thereof. In some cases, the targeting moieties disclosed herein bind to Cadm4. In some cases, the targeting moieties disclosed herein bind to the IgV set domain of Cadm4 or a portion thereof. In some cases, the targeting moieties disclosed herein bind to the IgC1 set domain of Cadm4 or a portion thereof. In some cases, the targeting moieties disclosed herein bind to the IgI set domain of Cadm4 or a portion thereof.

[0028] In some cases, the targeting moiety disclosed herein is an antibody or an antigen-binding fragment thereof that binds to a cell adhesion molecule (Cadm). In some cases, the targeting moiety disclosed herein is an antibody or an antigen-binding fragment thereof that binds to Cadm1. In some cases, the targeting moiety disclosed herein is an antibody or an antigen-binding fragment thereof that binds to the IgV set domain of Cadm1 or a portion thereof. In some cases, the targeting moiety disclosed herein is an antibody or an antigen-binding fragment that binds to the IgC1 set domain of Cadm1 or a portion thereof. In some cases, the targeting moiety disclosed herein is an antibody or an antigen-binding fragment thereof that binds to the IgI set domain of Cadm1 or a portion thereof. In certain cases, the targeting moiety disclosed herein is an antibody or an antigen-binding fragment thereof that binds to Cadm2. In some cases, the targeting moiety disclosed herein is an antibody or an antigen-binding fragment thereof that binds to the IgV set domain of Cadm2 or a portion thereof. In some cases, the targeting moiety disclosed herein is an antibody or an antigen-binding fragment thereof that binds to the IgC1 set domain of Cadm2 or a portion thereof. In some cases, the targeting moiety disclosed herein is an antibody or an antigen-binding fragment thereof that binds to the IgI set domain of Cadm2 or a portion thereof. In some cases, the targeting moiety disclosed herein is an antibody or an antigen-binding fragment thereof that binds to Cadm3. In some cases, the targeting moiety disclosed herein is an antibody or an antigen-binding fragment thereof that binds to the IgV set domain of Cadm3 or a portion thereof. In some cases, the targeting moiety disclosed herein is an antibody or an antigen-binding fragment thereof that binds to the IgC1 set domain of Cadm3 or a portion thereof. In some cases, the targeting moiety disclosed herein is an antibody or an antigen-binding fragment thereof that binds to the IgI set domain of Cadm3 or a portion thereof. In some cases, the targeting moiety disclosed herein is an antibody or an antigen-binding fragment thereof that binds to Cadm4.In some cases, the targeting moiety disclosed herein is an antibody or an antigen-binding fragment thereof that binds to the IgV set domain of Cadm4 or a portion thereof. In some cases, the targeting moiety disclosed herein is an antibody or an antigen-binding fragment thereof that binds to the IgC1 set domain of Cadm4 or a portion thereof. In some cases, the targeting moiety disclosed herein is an antibody or an antigen-binding fragment thereof that binds to the IgI set domain of Cadm4 or a portion thereof.

[0029] In some cases, the targeting moiety disclosed herein is a commercially available antibody that binds to Cadm1. In some cases, the targeting moiety disclosed herein is a commercially available antibody that binds to Cadm2. In some cases, the targeting moiety disclosed herein is a commercially available antibody that binds to Cadm3. In some cases, the targeting moiety disclosed herein is a commercially available antibody that binds to Cadm4. In some cases, the targeting moiety disclosed herein is anti-Cadm4 mAb244 / 5 (NeuroMAB). In some cases, the targeting moiety disclosed herein is the Necl4-Fc antibody of Eshed et al., Neuron, Vol. 47, No. 2, pp. 215-229. In some cases, the targeting moiety disclosed herein is an anti-SynCAM4 antibody (Biolegand, #833302). In some cases, the targeting moiety disclosed herein is an IGSF4C / SynCAM4 antibody (mdsystems, #MAB41642).

[0030] In some cases, the targeting moiety induces the complex to Schwann cells, and the complex is internalized when 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 Schwann cells. In some cases, the targeting moiety induces the complex to Schwann cells, and the complex is internalized when 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 Schwann cells. In some cases, the targeting moieties disclosed herein induce and internalize the complexes disclosed herein 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 or more after contacting Schwann cells. In some cases, the targeting moieties disclosed herein induce and internalize the complexes disclosed herein less than 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 9(3), 94, 95 or 96 hours after contact with Schwann cells.

[0031] In some cases, the drug molecules disclosed herein are released after being internalized into Schwann cells. In some cases, the drug molecules disclosed herein are released after the complexes disclosed herein enter the lysosomes of Schwann cells. In some cases, the drug molecules disclosed herein are released into the cytoplasm of Schwann cells. In some cases, the drug molecules disclosed herein are released into the nucleus of Schwann cells.

[0032] In some embodiments, the targeting moiety disclosed herein is an antibody or an antigen-binding fragment thereof. In some cases, the antibody or antigen-binding fragment thereof includes monovalent Fab’, bivalent Fab2, single-chain variable fragment (scFv), diabody, minibody, nanobody, single-domain antibody (sdAb) or camelid antibody or a binding fragment thereof. In other cases, the targeting moiety disclosed herein is (scFv)2, Fab, Fab’, F(ab’)2, Fv, dAb, Fd fragment, diabody, F(ab’)3, disulfide-bonded Fv, sdAb (VHH or nanobody), CDR, di-scFv, bi-scFv, tascFv (tandem scFv), tribody, tetrabody, V-NAR domain, Fcab, IgGACH2, DVD-Ig, probody, DARPin, Centyrin, affibody, affilin, afitin, anticalin, avimer, Fynomer, knotted domain peptide, monobody (or adnectin), tribody as well as nanofitin and miniprotein. In other cases, the targeting moiety disclosed herein is an antibody variant and derivative that includes an antibody functional fragment that retains the ability to bind to a receptor expressed on Schwann cells.Examples of functional fragments include Fab fragments (e.g., antibody fragments containing an antigen-binding domain and including portions of the light and heavy chains cross-linked by disulfide bonds); Fab’ (e.g., antibody fragments containing a single antigen-binding domain and including an additional portion of the heavy chain via the Fab’ and hinge region); F(ab’)2 (e.g., two Fab’ molecules linked by an interchain disulfide bond in the hinge region of the heavy chain; these Fab’ molecules can be directed against the same epitope or different epitopes); bispecific Fab (e.g., a Fab molecule having two antigen-binding domains, each of which can be directed against a different epitope); single-chain containing variable regions, also known as scFv (e.g., the variable antigen-binding determinant regions of a single light and heavy chain of an antibody linked to each other by a chain of 10 to 25 amino acids); disulfide-bonded Fv, or dsFv (e.g., the variable antigen-binding determinant regions of a single light and heavy chain of an antibody bound to each other by a disulfide bond); camelized VH (e.g., the variable antigen-binding determinant region of a single heavy chain of an antibody where some of the amino acids at the VH boundary are the amino acids identified in the heavy chain of a native camel 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); diabody (e.g., a dimerized scFv formed when the VH domain of a first scFv associates with the VL domain of a second scFv and the VL domain of the first scFv associates with the VH domain of the second scFv; the two antigen-binding regions of the diabody can be directed against the same epitope or different epitopes); triabody (e.g., a trimerized scFv that is formed in a similar manner to a diabody but has three antigen-binding domains, where the three antigen-binding domains occur in a single complex; the three antigen-binding domains can be directed against the same epitope or different epitopes); and tetrabody (e.g., a tetramerized scFv that is formed in a similar manner to a diabody but has four antigen-binding domains, where the four antigen-binding domains occur in a single complex; the four antigen-binding domains can be directed against the same epitope or different epitopes).In some embodiments, the targeting moiety disclosed herein is an antibody or an antigen-binding fragment thereof having monospecificity, bispecificity, trispecificity, or multispecificity. In other embodiments, the targeting moiety disclosed herein is one or more combinations of the antibodies or antigen-binding fragments thereof described above.

[0033] In some embodiments, the targeting moiety disclosed herein is a ligand molecule. In some cases, the ligand molecule is recognized by Schwann cells, and the complex disclosed herein is internalized within Schwann cells. In some cases, the ligand molecule includes a peptide, glycoprotein, carbohydrate moiety, dendrimer, or synthetic small molecule. In some cases, the ligand molecule disclosed herein binds to laminin 2. In some cases, the ligand molecule disclosed herein binds to the P0 protein. In some cases, the ligand molecule disclosed herein binds to gliomedin. In some cases, the ligand molecule disclosed herein binds to Cadm1. In some cases, the ligand molecule disclosed herein binds to Cadm2. In some cases, the ligand molecule disclosed herein binds to Cadm3. In some cases, the ligand molecule disclosed herein binds to Cadm4.

[0034] In other embodiments, the targeting moiety disclosed herein is one or more combinations of the antibodies, antigen-binding fragments thereof, or ligand molecules described above.

[0035] In some cases, the targeting moiety disclosed herein includes at least a portion of neurofascin-186 (NF186) or a derivative thereof. In some cases, the targeting moiety disclosed herein includes one or more gliomedin-binding domains of NF186.

[0036] In some cases, the targeted portion 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 gliomedin. In some cases, the targeted portion disclosed herein comprises 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or less of the full-length NF186 without significantly affecting the binding affinity to gliomedin. In some cases, the targeted portion disclosed herein comprises a portion of NF186 having one or more conservative amino acid substitutions without significantly affecting the binding affinity to gliomedin. In some cases, the targeted portion disclosed herein comprises a portion of NF186 having one or more non-conservative amino acid substitutions without significantly affecting the binding affinity to gliomedin. In some cases, the targeted portion disclosed herein comprises a portion of NF186 having one or more substitutions with basic, acidic, aromatic, aliphatic, acid amide, cyclic or sulfur-containing amino acids without significantly affecting the binding affinity to gliomedin. In some cases, the targeted portion disclosed herein comprises a portion of NF186 having one or more substitutions with standard or non-standard amino acids without significantly affecting the binding affinity to gliomedin. In some cases, the targeted portion disclosed herein comprises a portion of NF186 having one or more substitutions with alpha, beta, gamma or delta amino acids without significantly affecting the binding affinity to gliomedin. In some cases, the targeted portion 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 gliomedin. In some cases, the targeted portion 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 gliomedin. In some cases, the targeted portion disclosed herein comprises a portion of NF186 having one or more amino acid deletions without significantly affecting the binding affinity to gliomedin.In some cases, the targeting moiety disclosed herein comprises a portion of NF186 having one or more amino acid insertions without significantly affecting the binding affinity to gliomedin. In some cases, the targeting moiety disclosed herein comprises a portion of NF186 and an amino acid sequence that is at least partially reverse of the amino acid sequence of the corresponding portion of NF186 without significantly affecting the binding affinity to gliomedin. In some cases, the targeting moiety disclosed herein comprises a portion of NF186 without significantly affecting the binding affinity to gliomedin, wherein at least one of the amino acids of the original portion of NF186 is replaced with a stereoisomer (e.g., D stereoisomer) of that amino acid. In some cases, the targeting moiety disclosed herein comprises a portion of NF186 without significantly affecting the binding affinity to gliomedin and is extended at one or both ends by one or more groups such as D-amino acids. In some cases, the targeting moiety disclosed herein comprises NF186 having one or more combinations of the above-described modifications without significantly affecting the binding affinity to gliomedin.

[0037] In some cases, the targeting moiety disclosed herein comprises a recombinant NF186 peptide or a derivative thereof. In some cases, the targeting moiety disclosed herein comprises the gliomedin-binding domain of recombinant NF186. In some cases, the targeting moiety disclosed herein comprises at least 1%, 5%, 10%, 20%, 30%, 40%, 50% or 60% of full-length recombinant NF186 without significantly affecting the binding affinity to gliomedin. In some cases, the targeting moiety disclosed herein comprises 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or less of full-length recombinant NF186 without significantly affecting the binding affinity to gliomedin. In some cases, the targeting moiety disclosed herein comprises a portion of recombinant NF186 having one or more conservative amino acid substitutions without significantly affecting the binding affinity to gliomedin. In some cases, the targeting moiety disclosed herein comprises a portion of recombinant NF186 having one or more non-conservative amino acid substitutions without significantly affecting the binding affinity to gliomedin. In some cases, the targeting moiety disclosed herein comprises a portion of recombinant NF186 having one or more substitutions with basic, acidic, aromatic, aliphatic, acid amide, cyclic or sulfur-containing amino acids without significantly affecting the binding affinity to gliomedin. In some cases, the targeting moiety disclosed herein comprises a portion of recombinant NF186 having one or more substitutions with standard or non-standard amino acids without significantly affecting the binding affinity to gliomedin. In some cases, the targeting moiety disclosed herein comprises a portion of recombinant NF186 having one or more substitutions with alpha, beta, gamma or delta amino acids without significantly affecting the binding affinity to gliomedin. In some cases, 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 gliomedin.In some cases, the targeted portion 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 gliomedin. In some cases, the targeted portion disclosed herein comprises a portion of recombinant NF186 having one or more amino acid deletions without significantly affecting the binding affinity to gliomedin. In some cases, the targeted portion disclosed herein comprises a portion of recombinant NF186 having one or more amino acid insertions without significantly affecting the binding affinity to gliomedin. In some cases, the targeted portion disclosed herein comprises a portion of recombinant NF186 and an amino acid sequence that is at least partially reverse of the amino acid sequence of the corresponding portion of recombinant NF186 without significantly affecting the binding affinity to gliomedin. In some cases, the targeted portion disclosed herein comprises a portion of recombinant NF186 without significantly affecting the binding affinity to gliomedin, wherein at least one of the amino acids of the original portion of recombinant NF186 is replaced with a stereoisomer of that amino acid (e.g., D-stereoisomer). In some cases, the targeted portion disclosed herein comprises a portion of recombinant NF186 without significantly affecting the binding affinity to gliomedin, and is extended at one or both ends by one or more groups such as D-amino acids. In some cases, the targeted portion disclosed herein comprises recombinant NF186 having one or more combinations of the above-described modifications without significantly affecting the binding affinity to gliomedin.

[0038] In some cases, the targeting moiety disclosed herein binds to a nerve-specific extracellular matrix protein that binds to a receptor expressed on Schwann cells. In some cases, the targeting moiety disclosed herein binds to laminin 2. In some cases, the targeting moiety disclosed herein binds to the α2 subunit of laminin 2. In some cases, the targeting moiety disclosed herein binds to the G domain of the α2 subunit of laminin 2. In some cases, the targeting moiety disclosed herein binds to the G1 domain of the α2 subunit of laminin 2. In some cases, the targeting moiety disclosed herein binds to the G2 domain of the α2 subunit of laminin 2. In some cases, the targeting moiety disclosed herein binds to the G3 domain of the α2 subunit of laminin 2. In some cases, the targeting moiety disclosed herein binds to the G4 domain of the α2 subunit of laminin 2. In some cases, the targeting moiety disclosed herein binds to the G5 domain of the α2 subunit of laminin 2. In some cases, the targeting moiety disclosed herein binds to the β1 subunit of laminin 2. In some cases, the targeting moiety disclosed herein binds to the γ1 subunit of laminin 2.

[0039] In some cases, the targeting moiety disclosed herein comprises at least a portion of the glycolipid PGL-1 or a derivative thereof. In some cases, the targeting moiety disclosed herein comprises the unique trisaccharide of PGL-1 (in which 3,6-di-O-methylglucose is linked α-1→4 to 2,3-di-O-methyll-rhamnose and β-1→2 to 3-O-methyll-rhamnose).

[0040] In some cases, the targeting moiety disclosed herein comprises at least a portion of ML-LBP21 (also known as histone-like protein / Hlp) or a derivative thereof. In some cases, the targeting moiety disclosed herein comprises the laminin-2 binding domain of ML-LBP21. In some cases, the targeting moiety disclosed herein comprises at least 1%, 5%, 10%, 20%, 30%, 40%, 50% or 60% of full-length ML-LBP21 without significantly affecting the binding affinity to laminin-2. In some cases, the targeting moiety disclosed herein comprises 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or less of full-length ML-LBP21 without significantly affecting the binding affinity to laminin-2. In some cases, 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 cases, 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 cases, the targeting moiety disclosed herein comprises a portion of ML-LBP21 having one or more substitutions with basic, acidic, aromatic, aliphatic, acid amide, cyclic or sulfur-containing amino acids without significantly affecting the binding affinity to laminin-2. In some cases, the targeting moiety disclosed herein comprises a portion of ML-LBP21 having one or more substitutions with standard or non-standard amino acids without significantly affecting the binding affinity to laminin-2. In some cases, the targeting moiety disclosed herein comprises a portion of ML-LBP21 having one or more substitutions with alpha, beta, gamma or delta amino acids without significantly affecting the binding affinity to laminin-2. In some cases, 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 cases, the targeting moiety disclosed herein comprises a portion of ML-LBP21 having one or more substitutions with polar or nonpolar amino acids without significantly affecting the binding affinity to laminin-2. In some cases, 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 cases, 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 cases, the targeting moiety disclosed herein comprises a portion of ML-LBP21 and an amino acid sequence that is at least partially reverse of the amino acid sequence of the corresponding portion of ML-LBP21 without significantly affecting the binding affinity to laminin-2. In some cases, the targeting moiety disclosed herein comprises a portion of ML-LBP21 without significantly affecting the binding affinity to laminin-2, wherein at least one of the amino acids of the original portion of ML-LBP21 is replaced with a stereoisomer of that amino acid (e.g., D-stereoisomer). In some cases, the targeting moiety disclosed herein comprises a portion of ML-LBP21 without significantly affecting the binding affinity to laminin-2 and is extended at one or both ends with one or more groups such as D-amino acids. In some cases, the targeting moiety disclosed herein comprises ML-LBP21 combined with one or more of the above modifications without significantly affecting the binding affinity to laminin-2.

[0041] In some cases, the targeting moiety disclosed herein comprises a recombinant ML-LBP21 peptide or a derivative thereof. In some cases, the targeting moiety disclosed herein comprises the laminin-2 binding domain of recombinant ML-LBP21. In some cases, the targeting moiety disclosed herein comprises at least 1%, 5%, 10%, 20%, 30%, 40%, 50% or 60% of full-length recombinant ML-LBP21 without significantly affecting the binding affinity to laminin-2. In some cases, the targeting moiety disclosed herein comprises 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or less of full-length recombinant ML-LBP21 without significantly affecting the binding affinity to laminin-2. In some cases, the targeting moiety disclosed herein comprises a portion of recombinant ML-LBP21 having one or more conservative amino acid substitutions without significantly affecting the binding affinity to laminin-2. In some cases, 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 cases, the targeting moiety disclosed herein comprises a portion of recombinant ML-LBP21 having one or more substitutions with basic, acidic, aromatic, aliphatic, acid amide, cyclic or sulfur-containing amino acids without significantly affecting the binding affinity to laminin-2. In some cases, the targeting moiety disclosed herein comprises a portion of recombinant ML-LBP21 having one or more substitutions with standard or non-standard amino acids without significantly affecting the binding affinity to laminin-2. In some cases, the targeting moiety disclosed herein comprises a portion of recombinant ML-LBP21 having one or more substitutions with alpha, beta, gamma or delta amino acids without significantly affecting the binding affinity to laminin-2. In some cases, 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 cases, the targeting moiety disclosed herein comprises a portion of recombinant ML-LBP21 having one or more substitutions with polar or nonpolar amino acids without significantly affecting the binding affinity to laminin-2. In some cases, the targeting moiety disclosed herein comprises a portion of recombinant ML-LBP21 having one or more amino acid deletions without significantly affecting the binding affinity to laminin-2. In some cases, 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 cases, the targeting moiety disclosed herein comprises a portion of recombinant ML-LBP21 and an amino acid sequence that is at least partially reverse of the amino acid sequence of the corresponding portion of recombinant ML-LBP21 without significantly affecting the binding affinity to laminin-2. In some cases, the targeting moiety disclosed herein comprises a portion of recombinant ML-LBP21, wherein at least one of the amino acids of the original portion of recombinant ML-LBP21 is replaced with a stereoisomer (e.g., D-stereoisomer) of that amino acid without significantly affecting the binding affinity to laminin-2. In some cases, the targeting moiety disclosed herein comprises a portion of recombinant ML-LBP21 and is extended at one or both ends by one or more groups such as D-amino acids without significantly affecting the binding affinity to laminin-2. In some cases, the targeting moiety disclosed herein comprises recombinant ML-LBP21 having one or more combinations of the above-described modifications without significantly affecting the binding affinity to laminin-2.

[0042] In some embodiments, the targeting moiety disclosed herein comprises one or more combinations of the above-described molecules.

[0043] Drug molecule In some embodiments, the drug molecules disclosed herein alter the expression of disease-related molecules. In certain embodiments, overexpression of disease-related molecules causes disease. Thus, in some cases, the drug molecules disclosed herein reduce 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%. Thus, in some cases, the drug molecules disclosed herein reduce the transcription / RNA level of disease-related molecules to the level of a healthy individual. Thus, in other cases, 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%. Thus, in some cases, the drug molecules disclosed herein reduce the translation / protein level of disease-related molecules to the level of a healthy individual. In certain embodiments, the absence of disease-related molecules causes disease. Thus, in some cases, 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%. Thus, in some cases, the drug molecules disclosed herein increase the transcription / RNA level of disease-related molecules to the level of a healthy individual. Thus, in other cases, 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%. Thus, in some cases, the drug molecules disclosed herein increase the translation / protein level of disease-related molecules to the level of a healthy individual.

[0044] In other aspects, the drug molecules disclosed herein modify the activity of disease-related molecules. In certain aspects, the overactivity of disease-related molecules causes disease. Thus, in some cases, 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%. Thus, in some cases, the drug molecules disclosed herein reduce the activity of disease-related molecules to the level of a healthy individual. In certain aspects, the decrease in the activity of disease-related molecules causes disease. Thus, in some cases, 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%. Thus, in some cases, the drug molecules disclosed herein increase the activity of disease-related molecules to the level of a healthy individual.

[0045] In some cases, the drug molecule includes a drug or a prodrug form of the drug (drug precursor or prodrug). In some cases, the drug molecule includes a peptide. In some cases, the drug molecule includes a protein. Any suitable peptide or protein can be used as a drug molecule for altering the expression or activity of a disease-related molecule as disclosed herein. In some cases, the drug molecule includes an enzyme. In some cases, the peptide or protein can be generated, synthesized, and / or derivatized using several methodologies, such as, for example, phage display peptide libraries, one-bead one-compound peptide libraries, directed evolution, or position-scanning synthetic peptide combinatorial libraries. In some cases, the drug molecule includes a peptide or protein of about 2 to 25 amino acids, about 2 to 20 amino acids, about 2 to 15 amino acids, about 2 to 10 amino acids, or about 2 to 5 amino acids. The peptide or protein can include natural amino acids such as cysteine, alanine, or unnatural or modified amino acids. Unnatural amino acids include 3-amino acids, homoamino 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 cases, the peptide can be linear. In other cases, the peptide can be cyclic, for example bicyclic.

[0046] In some cases, the drug molecule includes a small molecule. In some cases, the small molecule is a modulator of a disease-related molecule. In some cases, the small molecule is an agonist of a disease-related molecule. In some cases, the small molecule is an antagonist of a disease-related molecule. In some cases, the small molecule is an inhibitor of a disease-related molecule.

[0047] In some cases, the drug molecule comprises an oligonucleotide. In some cases, the oligonucleotide comprises a double-stranded RNAi molecule or a single-stranded antisense oligonucleotide. In certain cases, the drug molecule comprises small interfering RNA (siRNA), microRNA (miRNA), inhibitory double-stranded RNA (dsRNA), small or short hairpin RNA (shRNA), piwi-interacting RNA (piRNA), heterogeneous nuclear RNA (hnRNA), small nuclear RNA (snRNA), or enzymatically prepared siRNA (esiRNA) or a precursor thereof. In other cases, the drug molecule comprises a nucleic acid disclosed in Sandy et al., Mammalian RNAi: a practical guide, BioTechniques, Vol. 39, No. 2 REVIEW, which is incorporated herein by reference in its entirety.

[0048] In some cases, 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.

[0049] In some cases, the double-stranded RNAi molecule or single-stranded antisense oligonucleotide is from about 9 to about 40 nucleotides in length. In some cases, the double-stranded RNAi molecule or single-stranded antisense oligonucleotide is from about 9 to about 36 nucleotides in length. In some cases, the double-stranded RNAi molecule or single-stranded antisense oligonucleotide is from about 10 to about 30 nucleotides in length. In some cases, the double-stranded RNAi molecule or single-stranded antisense oligonucleotide is from about 15 to about 30 nucleotides in length. In some cases, the double-stranded RNAi molecule or single-stranded antisense oligonucleotide is from about 20 to about 30 nucleotides in length. In some cases, the double-stranded RNAi molecule or single-stranded antisense oligonucleotide is from about 10 to about 25 nucleotides in length. In some cases, the double-stranded RNAi molecule or single-stranded antisense oligonucleotide is from about 10 to about 20 nucleotides in length. In some cases, the double-stranded RNAi molecule or single-stranded antisense oligonucleotide is at least 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 nucleotides in length. In some cases, the double-stranded RNAi molecule or single-stranded antisense oligonucleotide is 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 nucleotides or less in length. In some cases, the double-stranded RNAi molecule or single-stranded antisense oligonucleotide is from 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 in length.

[0050] In some cases, the drug molecules disclosed herein are double-stranded RNAi molecules having a sense strand and an antisense strand. In some embodiments, the sense strand and the antisense strand disclosed herein are reverse complementary to each other and form a duplex having a 3' overhang on the antisense strand. In some embodiments, the sense strand and the antisense strand disclosed herein are reverse complementary to each other and form a duplex having a 5' overhang on the antisense strand. In some embodiments, the sense strand and the antisense strand disclosed herein are reverse complementary to each other and form a duplex having a 3' overhang on the sense strand. In some embodiments, the sense strand and the antisense strand disclosed herein are reverse complementary to each other and form a duplex having a 5' overhang on the sense strand.

[0051] In some cases, the double-stranded RNAi molecule or single-stranded antisense oligonucleotide comprises at least one modified 2'-modified nucleotide. In some cases, the double-stranded RNAi molecule or single-stranded antisense oligonucleotide comprises at least one modified internucleotide linkage.

[0052] In some cases, the double-stranded RNAi molecules or single-stranded antisense oligonucleotides disclosed herein comprise one or more sugar-modified nucleotides. In some specific cases, the sugar-modified nucleotide is a 2'-fluoro-modified nucleotide. In some specific cases, the sugar-modified nucleotide is a 2'-alkoxy-modified nucleotide (e.g., 2'-methoxy-modified nucleotide). In some specific cases, the sugar-modified nucleotide is a 2'-amino-modified nucleotide. In some specific cases, the sugar-modified nucleotide is a 2'-azido-modified nucleotide.

[0053] In some cases, the double-stranded RNAi molecules or single-stranded antisense oligonucleotides disclosed herein contain one or more backbone-modified nucleotides. In some specific cases, the modified backbone is methylphosphonate. In some specific cases, the modified backbone is phosphorothioate. In some specific cases, the modified backbone is guanidinopropylphosphoramidate. In some specific cases, the modified backbone is mesylphosphoramidate (MsPA) linkage. In some specific cases, the modified backbone is phosphorothioate, and the phosphorothioate is a stereochemically enhanced phosphorothioate.

[0054] In some cases, the double-stranded RNAi molecules or single-stranded antisense oligonucleotides disclosed herein contain one or more purine modifications. In some specific cases, the purine modification disclosed herein is 2,6-diaminopurine. In some specific cases, the purine modification disclosed herein is 3-deaza-adenine. In some specific cases, the purine modification disclosed herein is 7-deaza-guanine. In some specific cases, the purine modification disclosed herein is 8-azidoadenine.

[0055] In some cases, the double-stranded RNAi molecules or single-stranded antisense oligonucleotides disclosed herein contain one or more pyrimidine modifications. In some specific cases, the pyrimidine modification disclosed herein is 2-thio-thymidine. In some specific cases, the pyrimidine modification disclosed herein is 5-carboxamido-uracil. In some specific cases, the pyrimidine modification disclosed herein is 5-methyl-cytosine. In some specific cases, the pyrimidine modification disclosed herein is 5-ethynyluracil.

[0056] In some cases, the polynucleotide molecules disclosed herein include abasic substitutions. When a hybridized polynucleotide construct is being considered for use as an siRNA, it is desirable to reduce miRNA-like off-target effects. Inclusion of one or more (e.g., one or two) abasic substitutions within the hybridized polynucleotide construct can reduce or eliminate miRNA-like off-target effects because the abasic substitution lacks a nucleobase that can participate in base-pairing interactions and relieve steric hindrance. Thus, the double-stranded RNAi molecules or single-stranded antisense oligonucleotides disclosed herein can include one or more (e.g., one or two) abasic substitutions. In certain cases, the abasic substitution is at the fifth nucleotide from the 5' end of the antisense strand disclosed herein. The polynucleotide molecules disclosed herein can contain a strand that includes a seed region that includes a hypoxanthine nucleobase-containing nucleoside (e.g., inosine).

[0057] In some cases, the double-stranded RNAi molecules or single-stranded antisense oligonucleotides disclosed herein include one or more types of modifications as described above. Thus, in some cases, about 10% of the nucleotides of the double-stranded RNAi molecules or single-stranded antisense oligonucleotides disclosed herein are modified with one or more types of modifications as described above. In other cases, about 20% of the nucleotides of the double-stranded RNAi molecules or single-stranded antisense oligonucleotides disclosed herein are modified with one or more types of modifications as described above. In other cases, about 30% of the nucleotides of the double-stranded RNAi molecules or single-stranded antisense oligonucleotides disclosed herein are modified with one or more types of modifications as described above. In other cases, about 40% of the nucleotides of the double-stranded RNAi molecules or single-stranded antisense oligonucleotides disclosed herein are modified with one or more types of modifications as described above. In other cases, about 50% of the nucleotides of the double-stranded RNAi molecules or single-stranded antisense oligonucleotides disclosed herein are modified with one or more types of modifications as described above. In other cases, about 60% of the nucleotides of the double-stranded RNAi molecules or single-stranded antisense oligonucleotides disclosed herein are modified with one or more types of modifications as described above. In other cases, about 70% of the nucleotides of the double-stranded RNAi molecules or single-stranded antisense oligonucleotides disclosed herein are modified with one or more types of modifications as described above. In other cases, about 80% of the nucleotides of the double-stranded RNAi molecules or single-stranded antisense oligonucleotides disclosed herein are modified with one or more types of modifications as described above. In other cases, about 90% of the nucleotides of the double-stranded RNAi molecules or single-stranded antisense oligonucleotides disclosed herein are modified with one or more types of modifications as described above. In other cases, 100% of the nucleotides of the double-stranded RNAi molecules or single-stranded antisense oligonucleotides disclosed herein are modified with one or more types of modifications as described above.

[0058] In some cases, one or more types of modifications disclosed herein occur at different positions within the double-stranded RNAi molecules or single-stranded antisense oligonucleotides disclosed herein. In certain cases, one or more types of modifications disclosed herein occur in the seed region within the double-stranded RNAi molecules or single-stranded antisense oligonucleotides disclosed herein. In certain cases, one or more types of modifications disclosed herein occur at the 3’ end of the double-stranded RNAi molecules or single-stranded antisense oligonucleotides disclosed herein. In certain cases, one or more types of modifications disclosed herein occur at the 5’ end of the double-stranded RNAi molecules or single-stranded antisense oligonucleotides disclosed herein. In certain cases, one or more types of modifications described herein occur dispersed within the double-stranded RNAi molecules or single-stranded antisense oligonucleotides described herein. In certain cases, one or more types of modifications disclosed herein occur in clusters within the double-stranded RNAi molecules or single-stranded antisense oligonucleotides disclosed herein.

[0059] In some cases, the disease-related molecule is peripheral myelin protein 22 (PMP22). Thus, the drug molecules disclosed herein modify PMP22. In some specific cases, PMP22 is associated with Charcot-Marie-Tooth disease type 1A (CMT1A). CMT damages the peripheral nervous system, and various types of CMT result in impairments in electrical transmission to the muscles, which then become dysfunctional. Specifically, CMT1A, the most common type of CMT (55% - 60% of all CMT patients), usually begins in puberty with muscle weakness and atrophy in the lower limbs, followed by muscle weakness and a decrease in sensation (e.g., touch, heat, cold) in the hands later in life. This disease is a progressive disease, and the symptoms usually worsen after the age of 45. The worsening of symptoms includes a decrease in the ability to perform simple tasks, instability in walking, a tendency to get injured due to frequent falls, and a decrease in the quality of life. The genetic defect in most CMT1A patients is a 1.5 Mb duplication on the chromosome segment containing the PMP22 gene. An increase above the basal concentration of the PMP22 protein causes demyelination.

[0060] In some cases, 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 cases, the double-stranded RNAi molecule or single-stranded antisense oligonucleotide comprises a continuous sequence of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides that is complementary to the target region of the human PMP22 transcript with 1, 2, 3, 4 or 5 or fewer mismatches. In other cases, 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 that is complementary to the target region of the human PMP22 transcript with 1, 2, 3, 4, or 5 or fewer mismatches. In some instances, the target region of the human PMP22 transcript is within the 5' UTR of the human PMP22 transcript. In some instances, the target region of the human PMP22 transcript is within the 3' UTR of the human PMP22 transcript. In some instances, the target region of the human PMP22 transcript is within the coding region of the human PMP22 transcript. In some instances, the target region of the human PMP22 transcript is in exon 1 of the human PMP22 transcript. In some instances, the target region of the human PMP22 transcript is in exon 2 of the human PMP22 transcript. In some instances, the target region of the human PMP22 transcript is in exon 3 of the human PMP22 transcript. In some instances, the target region of the human PMP22 transcript is in exon 4 of the human PMP22 transcript. In some instances, the target region of the human PMP22 transcript is in exon 5 of the human PMP22 transcript. In some instances, the target region of the human PMP22 transcript is in exon 6 of the human PMP22 transcript.In some cases, the target region of the human PMP22 transcript is in exon 7 of the human PMP22 transcript. In some cases, the target region of the human PMP22 transcript product includes the intron portion of the pre-PMP22 transcript product. In some cases, the target region of the human PMP22 transcript product includes the exon-intron junction of the pre-PMP22 transcript product.

[0061] In some cases, the disease-related molecule is an anti-ganglioside antibody. Therefore, the drug molecules disclosed herein modify the anti-ganglioside antibody. In some cases, the anti-ganglioside antibody is associated with Guillain-Barré syndrome. Guillain-Barré syndrome is an immune-mediated neuropathy after infection and is caused by the autoimmune destruction of nerves in the peripheral nervous system. This syndrome includes muscle movement and muscle movement that conveys pain, temperature, and touch. As a result, muscle strength may decrease and sensation in the legs and / or arms may be lost. The anti-ganglioside antibody may be involved in the etiology of Guillain-Barré syndrome.

[0062] In some cases, this disease-related molecule is a subunit of the SWI / SNF protein complex encoded by the gene SMARCB1. In some cases, this disease-related molecule is the LZTR1 protein encoded by the LZTR1 gene. Therefore, the drug molecules disclosed herein modify the subunit of the SWI / SNF protein complex. Therefore, the drug molecules disclosed herein modify the LZTR1 protein. In some cases, the subunit of the SWI / SNF protein complex is associated with schwannomatosis. In some cases, the LZTR1 protein is associated with schwannomatosis. Schwannomatosis is a type of neurofibromatosis that causes multiple schwannomas.

[0063] In some cases, the drug molecule is an immunoglobulin and the related disease is chronic inflammatory demyelinating polyneuropathy (CIDP). CIDP is a rare peripheral neuropathy characterized by a gradually progressive weakness involving symmetric motor and sensory loss and loss of deep tendon reflexes. This is caused by damage to Schwann cells. Due to the gradual onset of CIDP, diagnosis can be delayed for months to years, resulting in limited and delayed responses to treatment and significant nerve damage. Most people may require long-term treatment. Nearly one-third of CIDP patients may become wheelchair-dependent if left untreated. Early detection and appropriate treatment are important to help patients avoid significant physical disabilities.

[0064] Binding of the target molecule and the drug molecule In certain cases, the targeting moiety disclosed herein can be bound or conjugated directly (e.g., via a covalent or non-covalent bond) to the drug molecule disclosed herein, or via a linker (e.g., a cleavable linker versus a non-cleavable linker, or a peptide linker versus a non-peptide linker), or via an amino acid or other functional group.

[0065] In some cases, the targeting moiety disclosed herein and the drug molecule disclosed herein are bound via non-covalent interaction. In certain cases, the non-covalent interaction includes biotin / avidin interaction. Examples of linking the targeting moiety and the drug molecule include, but are not limited to, the use of biotin and avidin or streptavidin (see, e.g., U.S. Patent No. 4,885,172), typical biotin / avidin alternatives (e.g., FITC / anti-FITC (see, e.g., Harmer and Samuel (1989) J. Immunol. Meth. 122(1):115 - 221), digoxigenin / anti-digoxigenin, etc.

[0066] In other cases, the targeting moiety disclosed herein and the drug molecule disclosed herein are linked via a covalent interaction. In certain cases, the targeting moiety disclosed herein and the drug molecule disclosed herein are linked via chemical conjugation. For example, conventional chemical conjugation using a bifunctional coupling agent such as glutaraldehyde, diimide ester, aromatic and aliphatic diisocyanate, bis-p-nitrophenyl ester of dicarboxylic acid, aromatic disulfonyl chloride, and bifunctional aryl halide such as 1,5-difluoro-2,4-dinitrobenzene; p,p'-difluoro m,m'-dinitrodiphenyl sulfone, sulfhydryl-reactive maleimide, etc.

[0067] In some cases, the chemical conjugation between the targeting moiety disclosed herein and the drug molecule disclosed herein is via a non-cleavable linker or a cleavable linker.

[0068] In some cases, the cleavable linker is generally cleavable only intracellularly and preferably stable extracellularly in an extracellular environment such as that of Schwann cells. In certain cases, the cleavable linker disclosed herein includes a chemically cleavable linker. In certain cases, the cleavable linker disclosed herein includes an enzymatically cleavable linker.

[0069] A number of various chemically cleavable linkers are known to those skilled in the art (see, for example, U.S. Patent Nos. 4,618,492; 4,542,225 and 4,625,014). In some specific cases, the chemically cleavable linker is a pH-sensitive linker. In some cases, the pH-sensitive linkers disclosed herein are readily degraded in a high pH environment. In some cases, the pH-sensitive linkers disclosed herein are readily degraded in a low pH environment. In some cases, the pH-sensitive linker can be cleaved at a pH in the range of 4 - 6. In some cases, the pH-sensitive linker contains a hydrazone or a cyclic acetal. In some cases, the pH-sensitive linker is cleaved within an endosome or a lysosome. In other specific cases, the chemically cleavable linker is a glutathione-sensitive linker. In some cases, the glutathione-sensitive linker is cleaved by a disulfide exchange reaction with intracellular glutathione species. In some cases, the disulfide moiety further contains 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, etc. Acid-labile linkers are designed to be stable at the pH levels encountered in the blood, but become unstable and degrade when exposed to the low pH environment within lysosomes. Acid-labile linkers contain a moiety selected from the group consisting of hydrazones, acetals, cis-aconitate amides, and silyl ethers. Acid-sensitive linkers include, but are not limited to, hydrazones, acetals, cis-aconitate-like amides, and silyl ethers (see, for example, Perez et al., (2013) Drug Discov. Today, pp. 1 - 13). Hydrazones are readily synthesized, with a plasma half-life of 183 hours at pH 7 and 4.4 hours at pH 5, indicating selective cleavability under acidic conditions as confirmed in lysosomes (see, for example, Doronina et al. (2013) Nat. Biotechnol. 21(7):778 - 784).A disulfide bridge is a cleavable linker that utilizes the reducing environment of cells (see, for example, Saito et al. (2013) Adv. Drug Deliv. Rev. 55(2):199-215). After internalization and degradation, the disulfide bridge can release the drug within the lysosome.

[0070] Enzymatically cleavable linkers are selected to be cleaved by enzymes (e.g., proteases). In some cases, protease-cleavable linkers are typically designed to be stable in blood / plasma but are rapidly cleaved by lysosomal enzymes in the lysosome. 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 causes 1,6-elimination of carbon dioxide and simultaneous release of the free drug in its parent amine form (see, for example, Burke et al. (2009) Bioconjug. Chem. 20(6):1242-1250). In some specific cases, the cleavable linker is a protease-sensitive linker. The protease-sensitive linkers disclosed herein may include sequences cleavable by lysosomal proteases and / or endosomal proteases. In some cases, protease-sensitive linkers typically contain peptide sequences and can 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 cases, protease-sensitive linkers contain natural amino acids such as cysteine, alanine, or unnatural or modified amino acids. Unnatural amino acids include 3-amino acids, homoamino acids, proline derivatives, 3-substituted alanine derivatives, linear core amino acids, N-methyl amino acids. In some cases, protease-sensitive linkers contain the dipeptide sequence of valine-citrulline or alanine-citrulline. In some cases, protease-sensitive linkers can be cleaved by lysosomal proteases such as cathepsin B and / or endosomal proteases.

[0071] In some cases, the targeting moiety and the drug molecule are covalently attached to each other via a non-cleavable linker. Generally, non-cleavable linkers are not readily degraded in the intracellular or physiological environment. In some cases, the non-cleavable linkers disclosed herein include an optionally substituted alkyl group, and the substitution may include halogen substitution, hydroxyl group substitution, oxygen species substitution, and other common substitutions. In some cases, the non-cleavable linkers disclosed herein include an optionally substituted alkyl, an optionally substituted alkylene, an optionally substituted arylene, heteroarylene, a peptide sequence containing at least one unnatural amino acid, a cleaved glycan, a sugar or an enzyme-non-degradable sugar, an azide, an alkyne-azide, a peptide sequence containing an LPXT sequence, a thioether, biotin, biphenyl, a repeating unit of polyethylene glycol or an equivalent compound, an acid ester, an acid amide, a sulfamide, and / or an alkoxy-amine linker. In some cases, sortase-mediated ligation is utilized to covalently attach a drug molecule containing an LPXT sequence to a targeting moiety containing (G). In other cases, sortase-mediated ligation is utilized to covalently attach a targeting moiety containing an LPXT sequence to a drug molecule containing a (G) sequence (see, e.g., Proft T. Sortase-mediated protein ligation: an emerging biotechnology tool for protein modification and immobilization. Biotechnol Lett, 2010, 32(1): 1-10, which is incorporated herein by reference in its entirety).In some cases, the non-cleavable linkers disclosed herein are substituted alkylene, optionally substituted alkenylene, optionally substituted alkynylene, optionally substituted cycloalkylene, optionally substituted cycloalkenylene, optionally substituted arylene, optionally substituted heteroarylene further comprising at least one heteroatom selected from N, O, and S; optionally substituted heterocyclylene further comprising at least one heteroatom selected from N, O, and S; imino, optionally substituted nitrogen species, optionally substituted oxygen species O, optionally substituted sulfur species, or poly(alkylene oxide), such as polyethylene oxide or polypropylene oxide.

[0072] In some cases, the linker disclosed herein is a peptide linker. In various instances, the peptide linker is relatively short, typically about 20 amino acids or less, about 15 amino acids or less, about 10 amino acids or less, about 8 amino acids or less, about 5 amino acids or less, about 3 amino acids or less, or a single amino acid. In some cases, the peptide linker disclosed herein contains an amino acid sequence that is cleavable by a protease. In some cases, the peptide linker disclosed herein contains an amino acid sequence that is cleavable by cathepsin. In some cases, the peptide linker disclosed herein contains the dipeptide valine-citrulline (Val-Cit) (see U.S. Patent No. 6,214,345, which is hereby incorporated by reference in its entirety) or Phe-Lys. A library of dipeptide linkers was screened by Debowchik and colleagues, and the rate of doxorubicin release by enzymatic hydrolysis was measured (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). From these, it was confirmed that Phe-Lys was cleaved most rapidly with a half-life of 8 minutes, followed by Val-Lys, which was cleaved with a half-life of 9 minutes with a narrow margin. In contrast, the half-life of Val-Cit was 240 minutes. It was also confirmed from these that the cleavage rate decreased by the removal of the PAB group, which is probably due to steric hindrance of enzyme binding. In another study, the potencies of auristatin derivative MMAE linked by the dipeptide linkers Phe-Lys and Val-Cit and similar hydrazone linkers were compared. The Val-Cit linker was proven to be more than 100 times more stable than the hydrazone linker in human plasma. Most importantly, the Phe-Lys linker was significantly less stable than Val-Cit in human plasma, which is the reason for the current popularity of this linker (see, e.g., Doronina et al. (2003) Nat. Biotechnol. 21(7):778-784).

[0073] 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 FK GGGGS GGGGS GGGGS, and GGGGS GGGGS GGGGS VA GGGGS GGGGS GGGGS.

[0074] In some cases, the linkers disclosed herein utilize PASYLATION™ technology, which is a genetic fusion with a structurally irregular polypeptide sequence composed of the amino acids Pro, Ala, and / or Ser. In other cases, the linkers disclosed herein are selected from those disclosed in patents such as U.S. Patent No. 10,961,287, which is hereby incorporated by reference in its entirety.

[0075] In some cases, the linkers disclosed herein are non-peptide linkers. Glucuronide linkers incorporate hydrophilic sugar groups that are cleaved by the lysosomal enzyme beta-glucuronidase. When the sugar is cleaved from the phenol backbone, the moiety conjugated by the self-destruction of the PAB group is released (see, e.g., Jeffrey et al. (2006) Bioconjug. Chem. 17(3):831-840). In some cases, the linkers disclosed herein are derived from natural multi-domain proteins (see, e.g., Chen et al. (2013) Adv Drug Deliv Rev. 65(10):1357-1369).

[0076] The targeting moieties disclosed herein can be attached to the drug molecules disclosed herein via specific functional groups or via specific reactions. In some cases, the linkers disclosed herein contain functional groups that are reactive with corresponding functional groups on the targeting moieties and / or the drug molecules disclosed herein. A bifunctional linker has one functional group that is reactive with a group on the targeting moiety disclosed herein and another reactive functional group on the drug molecule disclosed herein and can be used to form the desired complex. In some instances, a heterobifunctional linker contains two or more different reactive groups that react with sites on the targeting moiety and the drug molecule disclosed herein, respectively. For example, a heterobifunctional crosslinker such as cysteine can contain an amine-reactive group and a thiol-reactive group that can interact with an aldehyde on a derivatized peptide. Additional combinations of reactive groups suitable for heterobifunctional crosslinkers include, for example, amine-reactive and sulfhydryl-reactive groups; carbonyl-reactive and sulfhydryl-reactive groups; amine-reactive and photoreactive groups; sulfhydryl-reactive and photoreactive groups; carbonyl-reactive and photoreactive groups; carboxylate-reactive and photoreactive groups; and arginine-reactive and photoreactive groups. Such reactions and functional groups are illustrative and non-limiting. Examples of other suitable reactive groups include, but are not limited to, thiol (-SH), carboxylate (COOH), carboxyl (-COOH), carbonyl, amine (NH2), hydroxyl (-OH), aldehyde (-CHO), alcohol (ROH), ketone (R2CO), active hydrogen, ester, sulfhydryl (SH), phosphate (-PO3), or a photoreactive moiety. Examples of amine-reactive groups include, but are not limited to, isothiocyanate, isocyanate, acyl azide, NHS ester, sulfonyl chloride, aldehyde and glyoxal, epoxide and oxirane, carbonate, arylating agent, imido ester, carbodiimide, and anhydride, among others.Examples of the thiol-reactive group include, but are not limited to, haloacetyl and alkyl halide derivatives, maleimide, aziridine, acryloyl derivatives, arylating agents, and thiol-disulfide exchange reagents. Examples of the carboxylate reactive group include, but are not limited to, diazoalkane and diazoacetyl compounds, such as carbonyldiimidazole and carbodiimide. Examples of the hydroxyl reactive group include, but are not limited to, epoxide and oxirane, carbonyldiimidazole, oxidation with periodic acid, N,N'-disuccinimidyl carbonate or N-hydroxysuccinimidyl chloroformate, enzymatic oxidation, alkyl halide, and isocyanate. Examples of the aldehyde-reactive group and the ketone-reactive group include, but are not limited to, hydrazine derivatives for Schiff base formation or reductive amination. Examples of the active hydrogen-reactive group include, but are not limited to, diazonium derivatives for Mannich condensation and iodination reaction. Examples of the photoreactive group include, but are not limited to, aryl azide and halogenated aryl azide, benzophenone, diazo compounds, and diazirine derivatives.

[0077] In some cases, the linkers disclosed herein bind a targeting moiety to a drug molecule by a cycloaddition reaction between an azide and an alkyne to form a triazole, where the azide and alkyne can be located on the targeting moiety, drug molecule, or linker. In some cases, the alkyne can be a cyclic alkyne, such as cyclooctyne. In some cases, the alkyne can be bicyclononyne (also known as bicyclo[6.1.0]nonyne or BCN) or a substituted bicyclononyne. In some cases, the cyclooctane is as described in International Patent Application Publication No. WO 2011 / 136645, "Fused Cyclooctyne Compounds And Their Use In Metal free Click Reactions", published November 3, 2011. In some cases, the azide can be a sugar or carbohydrate molecule that constitutes the azide. In some cases, the azide can be 6-azido-6-deoxygalactose or 6-azido-N-acetylgalactosamine. In some cases, the sugar or carbohydrate molecule that constitutes the azide is as described in International Patent Application Publication No. WO 2016 / 170186, "Process For The Modification Of A Glycoprotein Using A Glycosyltransferase That Is Or Is Derived From A β(1,4)-N-Acetylgalactosaminyltransferase", published October 27, 2016.In some cases, the cycloaddition reaction between an azide and an alkyne to form a triazole, where the azide and alkyne can be located on a targeting moiety, a drug molecule, or a linker, is described in International Patent Application Publication No. WO 2014 / 065661, entitled "Modified antibody, antibody-conjugate and process for the preparation thereof", published on May 1, 2014, or International Patent Application Publication No. WO 2016 / 170186, entitled "Process For The Modification Of A Glycoprotein Using A Glycosyltransferase That Is Or Is Derived From A β(1,4)-N-Acetylgalactosaminyltransferase", published on October 27, 2016.

[0078] In some cases, the linker disclosed herein binds a targeting moiety to a drug molecule by a Diels-Alder reaction between a dienophile and a diene / heterodiene, where the dienophile and diene / heterodiene can be located on the targeting moiety, the drug molecule, or the linker. In some cases, the linker is bound to the targeting moiety and / or the drug molecule by other pericyclic reactions such as an ene reaction. In some cases, the linker disclosed herein is bound to the targeting moiety and / or the drug molecule by an amide, thioamide, or sulfonamide bond-forming reaction. In some cases, the linker disclosed herein binds a targeting moiety to a drug molecule by a condensation reaction, forming an oxime group, a hydrazone group, or a semicarbazide group that exists between the linker, the targeting moiety, and the drug molecule.

[0079] In some cases, the linker disclosed herein binds the targeting moiety to the drug molecule by a conjugate addition reaction between a nucleophile (e.g., an amine or hydroxyl group) and an electrophile (e.g., a carboxylic acid or aldehyde). In some cases, prior to the reaction between the linker and the targeting moiety or drug molecule, the nucleophile may be present on the linker and the electrophile may be present on the targeting moiety or drug molecule. In some cases, prior to the reaction between the linker and the targeting moiety or drug molecule, the electrophile may be present on the linker and the nucleophile may be present on the targeting moiety or drug molecule. In some cases, the electrophile can be an azide, a silicon center, a carbonyl, a carboxylic acid, an anhydride, an isocyanate, a thioisocyanate, a succinimidyl ester, a sulfosuccinimidyl ester, a maleimide, a halogenated alkyl, a suspected halogenated alkyl, an epoxide, an episulfide, an aziridine, an aryl, an activated phosphorus center and / or an activated sulfur center. In some cases, the nucleophile can be an optionally substituted alkene, an optionally substituted alkyne, an optionally substituted aryl, an optionally substituted heterocyclyl, a hydroxyl group, an amino group, an alkylamino group, an anilide group or a thiol group.

[0080] In some cases, the linker disclosed herein further comprises a spacer, such as a polyethylene glycol spacer or an acyl / carbamoyl sulfamide spacer, such as HydraSpace™. In some cases, the spacer is as described in Verkade, J.M.M. et al., "A Polar Sulfamide Spacer Significantly Enhances the Manufacturability, Stability, and Therapeutic Index of Antibody-Drug Conjugates", Antibodies, 2018, Vol. 7, No. 12, which is hereby incorporated by reference in its entirety.

[0081] Generally, the linkers disclosed herein do not have specific biological activities other than binding proteins or maintaining the minimum distance or other spatial relationships between proteins. However, the constituent amino acids of the spacer can be selected to affect the properties of the molecule, such as folding, net charge, and hydrophobicity. In certain cases, the linker may contain an enzyme cleavage site.

[0082] After chemical synthesis, biological expression, or purification, the fusion protein may have a conformation substantially different from the native conformation of the constituent polypeptides. In this case, it may be necessary to denature and reduce the polypeptide and then refold the polypeptide into the preferred conformation. Methods for reducing and denaturing proteins and methods for inducing refolding are well known to those skilled in the art (see, for example, 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).

[0083] In some cases, 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 nucleotide. In some cases, the peptide / protein or antibody or an antigen-binding fragment thereof contains a non-natural amino acid to which a nucleotide can covalently bind. In some cases, the peptide / protein or antibody or an antigen-binding fragment thereof is covalently bound to a nucleotide via conjugation to a lysine residue or a cysteine residue of the peptide / protein or antibody or an antigen-binding fragment thereof. In some cases, the nucleotide is conjugated to a cysteine residue of the peptide / protein or antibody or an antigen-binding fragment thereof via a maleimide-containing linker, and optionally, the maleimide-containing linker contains a maleimidocaproyl or maleimidomethylcyclohexane-1-carboxylate group. In some instances, the peptide / protein or antibody or an antigen-binding fragment thereof is glycosylated, which includes at least one sugar moiety to which a nucleotide can covalently bind. In some cases, the at least one sugar moiety includes at least one sugar moiety that is branched mannose. In some cases, the peptide / protein or antibody or an antigen-binding fragment thereof is glycosylated, which includes two or more sugar moieties, and all or some of each of the two or more sugar moieties is covalently bound to a different nucleotide. In some cases, the peptide / protein or antibody or an antigen-binding fragment thereof is fully glycosylated or partially glycosylated. The partial glycosylation can be generated via chemical means or enzymatic means. In some cases, the partial glycosylation is generated in cells deficient in an enzyme of the N- or O-glycosylation pathway. In some instances, the linkers disclosed herein are bound to the peptide / protein or antibody or an antigen-binding fragment thereof via a phosphate bond, a thioether bond, an ether bond, a carbon-carbon bond, or an amide bond. In some instances, the linkers disclosed herein are bound to a nucleotide via a phosphate group or a phosphorothioate group, such as a terminal phosphate of an oligonucleotide backbone.

[0084] In some specific cases, 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. Thus, in some cases, the targeting moiety disclosed herein is conjugated to the 3'-end of the sense strand. In some cases, the targeting moiety disclosed herein is conjugated to the 5'-end of the sense strand. In some cases, the targeting moiety disclosed herein is conjugated to the 3'-end of the antisense strand. In some cases, the targeting moiety disclosed herein is conjugated to the 5'-end of the antisense strand.

[0085] In some cases, both the targeting moiety and the drug molecule are a peptide / protein or an antibody or an antigen-binding fragment thereof. Thus, in some cases, the targeting moiety and the drug molecule can be expressed as a fusion protein. In some specific cases, the targeting moiety and the drug molecule are directly bound to each other. In some specific cases, the targeting moiety and the drug molecule are bound via intervening amino acids. In some specific cases, the targeting moiety and the drug molecule are bound via a peptide linker. Specifically, the linker can bind to the constituent amino acids via its side chain (e.g., via a disulfide bond to cysteine), but in other cases, the linker binds to the alpha-carbon amino and carboxyl groups of the terminal amino acids, if they are present.

[0086] To generate a fusion protein, generally, a DNA sequence encoding the fusion protein is prepared, the DNA is placed under the control of a specific promoter in an expression cassette, the protein is expressed in a host, the expressed protein is isolated, and the protein is refolded if necessary. The DNA encoding the fusion protein can be prepared by any suitable method, including cloning and restriction of appropriate sequences, or direct chemical synthesis by methods such as the phosphotriester method of Narang et al. (1979) Meth. Enzymol. 68:90-99; the phosphodiester method of Brown et al. (1979) Meth. Enzymol. 68:109-151; the diethylphosphoramidite method of Beaucage et al. (1981) Tetra. Lett., 22:1859-1862; and the solid support method of U.S. Patent No. 4,458,066. In some cases, the DNA encoding the fusion protein constructs disclosed herein can be cloned using DNA amplification methods such as polymerase chain reaction (PCR). Thus, for example, the nucleic acid sequence encoding the targeting moiety disclosed herein can be PCR amplified using primers containing engineered restriction sites. This generates a nucleic acid encoding the targeting moiety disclosed herein and having terminal restriction sites. Similarly, the nucleic acids encoding the drug molecules disclosed herein can be provided with complementary restriction sites. Ligation of the sequences and insertion into a vector generates a vector encoding the fusion protein. The nucleic acid sequence encoding the fusion protein can be expressed in a variety of host cells, including Escherichia coli (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 is operably linked to expression control sequences appropriate for each host. In the case of E. coli, this includes a promoter such as the T7, trp, or lambda promoter, a ribosome binding site, and preferably a transcription termination signal.In the case of eukaryotic cells, the control sequences include a promoter, preferably an enhancer and polyadenylation sequence derived from an immunoglobulin gene, SV40, cytomegalovirus, etc., and may include splice donor and acceptor sequences. The plasmid can be introduced into the selected host cell by well-known methods such as calcium chloride transformation in the case of E. coli, calcium phosphate treatment or electroporation in the case of mammalian cells. Cells transformed with the plasmid can be selected by the antibiotic resistance conferred by the genes contained in the plasmid, such as the amp, gpt, neo and hyg genes.

[0087] When expressed, the recombinant fusion protein can be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity column, column chromatography, gel electrophoresis, etc. (generally, see R. Scopes (1982) Protein Purification, Springer-Verlag, N.Y.; Deutscher (1990) Methods in Enzymology Vol. 182: Guide to Protein Purification, Academic Press, Inc. N.Y.). A substantially pure composition with at least about 90 - 95% homogeneity is preferred, and for pharmaceutical use, a homogeneity of 98 - 99% or more is most preferred. The polypeptide, purified partially or to the desired homogeneity, can be used for therapy.

[0088] In some cases, certain modifications can be added to the fusion protein without reducing its biological activity. Some modifications can be made to facilitate cloning, expression, or incorporation of the drug molecules or targeting moieties disclosed herein into the fusion protein. Such modifications include, for example, methionine added to the amino terminus to provide an initiation site, or additional amino acids placed at either terminus to generate conveniently located restriction sites or stop codons.

[0089] In some cases, one of the targeting moiety and the drug molecule is a peptide / protein or antibody or an antigen-binding fragment thereof, and the other is a small molecule. Exemplary linkers are described in U.S. Patent Nos. 10,864,279, 9,814,784, 11,173,214, 11,104,968, 9,872,924, 10,537,644, U.S. Patent Application Publication Nos. 2018 / 0169262, 2022 / 0072137, and 2018 / 0311375, which are hereby incorporated by reference in their entirety.

[0090] In some cases, 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. Thus, exemplary linkers are described in U.S. Patent Nos. 8,772,472, 9,211,343, 8,969,543, 9,074,208, 10,947,323, U.S. Patent Application Publication Nos. 2012 / 0177723, 2012 / 0108803, 2004 / 0053876, 2021 / 0108200, and 2015 / 0315587, which are hereby incorporated by reference in their entirety.

[0091] Other properties of the drug conjugate and pharmaceutical composition In certain cases, multiple targeting moieties disclosed herein can be bound to a single drug molecule disclosed herein. In certain cases, multiple drug molecules disclosed herein can be bound to a single targeting moiety disclosed herein. In certain cases, a single targeting moiety disclosed herein is bound to a single drug molecule disclosed herein. In some cases, the ratio of drug molecule to targeting moiety is 1:1. In some cases, the ratio of drug molecule to targeting moiety is about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. In some cases, the ratio of drug molecule to targeting moiety is about 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10.

[0092] In some embodiments, provided herein is a pharmaceutical composition comprising a drug conjugate disclosed herein and a pharmaceutically acceptable excipient. In some cases, the pharmaceutical composition is formulated for parenteral, intravenous, subcutaneous, intrathecal, or intraischial injection.

[0093] In some cases, the drug conjugate provided herein preferably internalizes into Schwann cells. In some cases, the drug conjugate provided herein internalizes into Schwann cells at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times more than into cell types other than Schwann cells.

[0094] In some cases, the drug conjugate provided herein enters the bloodstream and remains therein. In some cases, the drug conjugate provided herein enters the bloodstream and effectively passes through the blood-nerve barrier. In some cases, the drug conjugate provided herein enters the bloodstream, effectively passes through the blood-nerve barrier, and concentrates within the sciatic nerve.

[0095] The pharmaceutical compositions disclosed herein can be prepared to contain the drug complexes 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 proteins, gelatin, starch, vitamins, cellulose and its derivatives, animal and vegetable oils, polyethylene glycol and solvents such as sterile water, alcohol, glycerol, and polyhydric alcohols. Intravenous vehicles include infusion fluids and nutrient replenishers. Preservatives include antibacterial agents, antioxidants, chelating agents, and inert gases. Other pharmaceutically acceptable vehicles include, for example, non-toxic excipients containing aqueous solutions, salts, preservatives, buffers, etc. described in Remington: The Science and Practice of Pharmacy, 21st Edition, edited by Gennaro, Lippencott Williams & Wilkins (2005), and The United States Pharmacopeia: The National Formulary (USP 36 NF31) published in 2013. The pH and exact concentrations 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".

[0096] The pharmaceutical compositions disclosed herein can be administered locally or systemically. The therapeutically effective amount varies depending on factors such as the degree of infection of the subject, the individual's age, sex, and weight. The dosing regimen can be adjusted to obtain an optimal therapeutic effect. For example, it can be administered several times a day or the dose can be proportionally reduced in response to the urgency of the treatment situation.

[0097] The pharmaceutical composition can be administered by convenient methods such as injection (e.g., subcutaneous administration, intravenous administration, intraorbital administration, intrascapular administration, intrathecal administration, etc.), oral administration, ophthalmic application, inhalation, topical application, or rectal administration. Depending on the route of administration, the pharmaceutical composition can be coated with materials to protect it from the action of enzymes, acids, and other natural conditions that may inactivate the pharmaceutical composition. The pharmaceutical composition can also be administered parenterally or intraperitoneally. The dispersant can also be prepared in glycerol, liquid polyethylene glycol, and their mixtures, as well as in oils. Under normal storage and use conditions, these formulations may contain preservatives to prevent the growth of microorganisms.

[0098] Pharmaceutical compositions suitable for injection include sterile aqueous solutions (if water-soluble) or dispersions, and sterile powders for the immediate preparation of sterile injection solutions or dispersions. In some cases, the composition is typically sterile and has sufficient fluidity to be easily injectable. In some cases, the composition is stable under the conditions of manufacture and storage and is protected against the contaminating action of microorganisms such as bacteria and fungi. The medium can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. The appropriate fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, isotonic agents such as sugars, polyhydric alcohols such as mannitol, sorbitol, or sodium chloride are used in the composition. Prolongation of the absorption of the injectable composition is achieved, for example, by including in the composition agents that delay absorption such as aluminum monostearate and gelatin.

[0099] The sterile injectable solution can be prepared by incorporating the required amount of the pharmaceutical composition in a suitable solvent containing one or a combination of the ingredients listed above, as required, and then filtering and sterilizing. Generally, the dispersion is prepared by incorporating the pharmaceutical composition into a sterile vehicle containing a basic dispersion medium and the other required ingredients listed above.

[0100] In some cases, the pharmaceutical composition is formulated in unit dosage form for ease of administration and uniformity of dosage. As used herein, the unit dosage form refers to physically discrete units suitable as unit dosages for the subject to be treated. Each unit containing a predetermined amount of the pharmaceutical composition is calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle. The specification of the unit dosage form is related to the characteristics of the pharmaceutical composition and the particular therapeutic effect to be achieved. The principal pharmaceutical compositions are formulated in acceptable unit dosages, with a suitable pharmaceutically acceptable vehicle, for convenient and effective administration in effective amounts. In the case of compositions containing auxiliary active ingredients, the dosage is determined by reference to the usual dosage and method of administration of the ingredients.

[0101] The pharmaceutical composition can be administered orally, for example, with a carrier, in, for example, an enteric-coated unit dosage form. The pharmaceutical composition and other ingredients can also be encapsulated in hard-shell or soft-shell gelatin capsules or compressed into tablets. In oral therapeutic administration, the pharmaceutical composition is formulated with excipients and can be used in the form of ingestible tablets, troches, capsules, pills, wafers, etc. The percentages of the composition and preparation can of course vary and it is convenient for them to be between about 5% and about 80% of the unit weight. Tablets, troches, pills, capsules, etc. can also include the following: binders such as tragacanth, acacia, corn starch, gelatin; excipients such as dicalcium phosphate; disintegrants such as corn starch, potato starch, alginic acid; lubricants such as magnesium stearate; sweetening agents such as sucrose, lactose, saccharin or flavoring agents such as peppermint, wintergreen oil, cherry flavor. When the dosage unit form is a capsule, in addition to the above types of materials, a liquid carrier can be included. Various other materials can be present as coatings or to change the physical form of the dosage unit. For example, tablets, pills, capsules can be coated with shellac, sugar, or both. Syrups or elixirs can contain a drug, sucrose as a sweetening agent, methylparaben and propylparaben as preservatives, a dye, and a flavoring agent such as cherry flavor or orange flavor. All materials used in the preparation of the dosage unit form must be of pharmaceutically acceptable purity and substantially non-toxic in the amounts used. Further, the pharmaceutical composition can be formulated with sustained-release regulators and formulations.

[0102] Regarding the polynucleotide molecules disclosed in this specification, suitable pharmaceutically acceptable salts include: (i) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine; (ii) acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid; and (iii) salts formed with organic acids such as acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, etc.

[0103] In some cases, the drug molecules disclosed herein are encapsulated within a nanoparticle system that includes multiple particles such as, but not limited to, polymers, gold particles, silica, lipids, or protein-based particles, and the multiple particles are formulated to be conjugated with the targeting moieties disclosed herein (see International Publication No. WO 2023 / 056282, which is hereby incorporated by reference in its entirety). In some cases, the multiple particles are conjugated with the targeting moieties disclosed herein by non-covalent bonds (e.g., adsorption), and such conjugation maintains the biological activity of the targeting moieties. In some cases, the multiple particles are conjugated with the targeting moieties disclosed herein by covalent bonds, and such conjugation maintains the biological activity of the targeting moieties. In some cases, adsorption is a non-covalent immobilization technique such as, but not limited to, physical adsorption or ionic bonding. In some cases, physical adsorption involves the adhesion of the targeting moieties disclosed herein to the surface of the particles via weak interactions (e.g., electrostatic forces, hydrogen bonding forces, hydrophobic forces, or van der Waals forces). In some cases, ionic bonding is based on ionic bonds between the oppositely charged surfaces of the targeting moieties disclosed herein and the particles. In some cases, covalent bonding requires prior activation of the particles. In some cases, covalent bonding is based on carbodiimide chemistry, maleimide chemistry (see Jahan S.T et al., Int. J. Drug Deliv. 2017, which is hereby incorporated by reference in its entirety), or "click chemistry" (see Yi, G et al., Biomater. Res. 2018, which is hereby incorporated by reference in its entirety). In some specific cases, the targeting moiety includes an antibody or an antigen-binding fragment thereof disclosed herein and is conjugated to the nanoparticle system using maleimide-thiol chemistry (see Niels Dammes et al., Nat Nanotechnol. 2021, which is hereby incorporated by reference in its entirety).In certain specific cases, the targeting moieties disclosed herein are conjugated to the surface of a nanoparticle system comprising multiple polymer nanoparticles (see Nasrul Wathoni et al., Drug Delivery, 2022, which is hereby incorporated by reference in its entirety).

[0104] In some instances, the drug molecules disclosed herein are encapsulated in extracellular vesicles (EVs), which are modified to present the targeting moieties disclosed herein on their outer surface (see International Publication No. WO 2020 / 141369 or Chan, H. et al., Front. Cell. Dev. Biol., 2021, Vol. 9, p. 751079, which are hereby incorporated by reference in their entirety). In certain specific cases, the targeting moieties disclosed herein are tethered to the surface of EVs by fusion with lysosome-associated membrane glycoprotein 2b protein (see Liang, Y. et al., ACS Appl Mater Interfaces., 2020, Vol. 12, p. 36938, which is hereby incorporated by reference in its entirety). In certain specific cases, the targeting moieties disclosed herein are tethered to the surface of EVs via bio-orthogonal chemistry (see Tian, T. et al., Biomaterials, 2018, Vol. 150, p. 137, which is hereby incorporated by reference in its entirety).

[0105] Method In some embodiments, provided herein is a method for targeted delivery of a drug to Schwann cells by contacting a Schwann cell with a drug conjugate disclosed herein or a pharmaceutical composition comprising a drug conjugate disclosed herein. In some cases, the drug conjugate is internalized into Schwann cells when the targeting moiety binds to a cell surface molecule expressed on the Schwann cell. In some cases, the cell surface molecule expressed on the Schwann cell is a cell surface receptor having an extracellular domain. In some cases, the cell surface molecule is concentrated on the Schwann cell surface. In some cases, the drug conjugate is delivered to Schwann cells by systemic delivery of a drug conjugate disclosed herein or a pharmaceutical composition disclosed herein to the body of a subject. In some cases, a drug conjugate disclosed herein can pass through the blood-nerve barrier. In some cases, a drug conjugate disclosed herein can pass through the blood-nerve barrier and be concentrated in the sciatic nerve. In some embodiments, provided herein is a preferred method for delivery of a drug to or into Schwann cells by contacting a Schwann cell with a drug conjugate disclosed herein or a pharmaceutical composition comprising a drug disclosed herein. In some cases, the drug conjugate is preferably internalized into Schwann cells when the targeting moiety binds to a cell surface molecule expressed on the Schwann cell. In some cases, the cell surface molecule preferably expressed on the Schwann cell is a cell surface receptor having an extracellular domain. In some cases, the cell surface molecule is preferably expressed on the Schwann cell surface. In some cases, the cell surface molecule is expressed at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% less or 2, 3, 4, 5 times more in Schwann cells than in other cell types. In some cases, the method provided herein preferably ensures that the drug is internalized into Schwann cells at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% less or 2, 3, 4, 5, 6, 7, 8, 9 or 10 times more than into cell types other than Schwann cells.In some cases, the drug conjugate is delivered to Schwann cells by systemically delivering the drug conjugate disclosed herein or the pharmaceutical composition disclosed herein to the body of the subject. In some cases, the drug conjugate disclosed herein can pass through the blood-nerve barrier. In some cases, the drug conjugate disclosed herein can pass through the blood-nerve barrier and be concentrated in the sciatic nerve.

[0106] In some cases, the target delivery method disclosed herein has improved pharmacokinetics. In certain cases, the target delivery method disclosed herein results in a higher Cmax (i.e., the maximum concentration of drug molecules within Schwann cells). In certain cases, the target delivery method disclosed herein results in a shorter Tmax when reaching Cmax within Schwann cells. In certain cases, the target delivery method disclosed herein results in an increased area under the concentration-time curve. In certain cases, the target delivery method disclosed herein results in a longer 1 / 2 t (i.e., the time required for the concentration of drug molecules to decrease by half after reaching distribution equilibrium within Schwann cells). Thus, in some cases, the target delivery method disclosed herein results in the circulation and availability of the drug conjugate in the subject for at least 1, 5, 10, 15, 20, 25, 30, 35, or 40 days. In some cases, the target delivery method disclosed herein results in the circulation and availability of the drug conjugate in the subject for about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120 days. In some cases, the target delivery method disclosed herein results in the circulation and availability of the drug conjugate in the subject for up to 90, 120, 150, 180, 210, or 240 days.

[0107] In some cases, the target delivery methods disclosed herein result in a decrease in distribution in any tissue, organ, or cell type other than Schwann cells. In some cases, the target delivery methods disclosed herein result in only a small or minimal amount in any tissue, organ, or cell type other than Schwann cells. In some cases, the target delivery methods disclosed herein are non-immunogenic. In some cases, the target delivery methods disclosed herein are biodegradable. In some cases, the target delivery methods disclosed herein are applicable to various drug molecules.

[0108] In some cases, the target delivery method is achieved by parenteral, intravenous, subcutaneous, intrathecal, or intramuscular injection of the pharmaceutical composition disclosed herein. In some cases, the target delivery method is achieved by intravenous injection of the pharmaceutical composition disclosed herein. In some cases, the target delivery method is achieved by local delivery (see Pitiot et al. (2022) Antibodies (Basel 11(3):56).

[0109] In another aspect, a method of 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 or a pharmaceutical composition disclosed herein; and administering the drug conjugate or pharmaceutical composition to the subject to treat a peripheral demyelinating disease or peripheral neuropathy, or alleviate or reduce the symptoms of a peripheral demyelinating disease or peripheral neuropathy, is provided herein.

[0110] In some cases, the peripheral demyelinating disease or peripheral neuropathy is Charcot-Marie-Tooth disorder, Guillain-Barré syndrome (acute inflammatory demyelinating polyneuropathy type), neurofibroma, chronic inflammatory demyelinating polyneuropathy, nerve trauma, chemotherapy-induced neuropathy, diabetic peripheral neuropathy, migraine, schwannoma, neurofibromatosis type 1 (NF1), malignant peripheral nerve sheath tumor (MPNST), or nerve injury.

[0111] In some cases where an increase in a disease-related molecule causes the disease, the drug complex or pharmaceutical composition is administered in a dosage and schedule effective to reduce the expression of the disease-related 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 cases where an increase in a disease-related molecule causes the disease, the drug complex or pharmaceutical composition is administered in a dosage and schedule effective to reduce the expression of the disease-related 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 cases where an increase in a disease-related molecule causes the disease, the drug complex or pharmaceutical composition is administered in a dosage and schedule effective to reduce the activity of the disease-related 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 cases where an increase in a disease-related molecule causes the disease, the drug complex or pharmaceutical composition is administered in a dosage and schedule effective to reduce the activity of the disease-related molecule by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%.

[0112] In some cases, the drug conjugate or pharmaceutical composition is administered at a dose and schedule effective to reduce PMP22 expression by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%. In some cases, the drug conjugate or pharmaceutical composition is administered at a dose and schedule effective to reduce PMP22 expression by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%. In some cases, the drug conjugate or pharmaceutical composition is administered at a dose and schedule effective to reduce PMP22 activity by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%. In some cases, the drug conjugate or pharmaceutical composition is administered at a dose and schedule effective to reduce PMP22 activity by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%.

[0113] In some cases, the drug conjugate or pharmaceutical composition is administered at a dosage and schedule effective to reduce the expression 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 cases, the drug conjugate or pharmaceutical composition is administered at a dosage and schedule effective to reduce the expression 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%. In some cases, the drug conjugate or pharmaceutical composition is administered at a dosage 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 cases, the drug conjugate or pharmaceutical composition is administered at a dosage 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%.

[0114] In some cases where the absence of a disease-related molecule causes the disease, the drug conjugate or pharmaceutical composition is administered in a dosage and schedule effective to increase the expression of the disease-related 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 cases where the absence of a disease-related molecule causes the disease, the drug conjugate or pharmaceutical composition is administered in a dosage and schedule effective to increase the expression of the disease-related 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 cases where the absence of a disease-related molecule causes the disease, the drug conjugate or pharmaceutical composition is administered in a dosage and schedule effective to increase the activity of the disease-related 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 cases where the absence of a disease-related molecule causes the disease, the drug conjugate or pharmaceutical composition is administered in a dosage and schedule effective to increase the activity of the disease-related molecule by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%.

[0115] In some cases, the drug conjugate or pharmaceutical composition is administered at a dose and schedule effective to increase the expression of the 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 cases, the drug conjugate or pharmaceutical composition is administered at a dose and schedule effective to increase the expression of the 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 cases, the drug conjugate or pharmaceutical composition is administered at a dose and schedule effective to reduce the activity of the 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 cases, the drug conjugate or pharmaceutical composition is administered at a dose and schedule effective to reduce the activity of the 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%.

[0116] In some cases, 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 cases, 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 cases, 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 cases, 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%.

[0117] In some cases, the drug conjugate or pharmaceutical composition is administered at a dose and schedule effective to increase the expression of a myelination marker gene 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 are present at different developmental or wound repair stages and express different Schwann cell-specific markers. Thus, in certain cases, the drug conjugate or pharmaceutical composition is administered at a dose and schedule effective to increase the expression of a Schwann cell progenitor cell marker gene 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 cases, the drug conjugate or pharmaceutical composition is administered at a dose and schedule effective to increase the expression of an immature Schwann cell marker gene 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 cases, the drug conjugate or pharmaceutical composition is administered at a dose and schedule effective to increase the expression of a myelinated Schwann cell marker gene 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 cases, the drug conjugate or pharmaceutical composition is administered at a dose and schedule effective to increase the expression of a non-myelinated Schwann cell marker gene by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%. In some cases, the drug conjugate or pharmaceutical composition includes, but is not limited to, S100, p75 NTRAdministered in an effective dose and schedule to increase the expression of one or more markers, including 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%.

[0118] Certain terms As used herein, the term "disease-related molecule" generally refers to a molecule that is directly or indirectly related to the onset, progression, prolongation, or recurrence of a disease. In some cases, abnormal expression levels of disease-related molecules are associated with the onset, progression, prolongation, or recurrence of a disease. In some cases, abnormal activities of disease-related molecules are associated with the onset, progression, prolongation, or recurrence of a disease.

[0119] As used herein, the term "complementary" generally refers to the ability of two nucleotides or two sets of nucleotides to pair precisely. In particular, complementary refers to a term that characterizes the degree of hydrogen bond pairing that results in a bond between two nucleotides or two sets of nucleotides. For example, if a base at a certain position of an oligonucleotide can hydrogen bond with the base at the corresponding position of a target nucleic acid (such as mRNA), the bases are considered to be complementary to each other at that position. Base pairing can include both normal Watson-Crick base pairing and non-Watson-Crick base pairing (such as wobble base pairing and Hoogsteen base pairing). For example, in some cases, for complementary base pairing, an adenosine-type base (A) is complementary to a thymidine-type base (T) or a uracil-type base (U), a cytosine-type base (C) is complementary to a guanosine-type base (G), and universal bases such as 3-nitropyrrole or 5-nitroindole can hybridize with any A, C, U, or T and are considered to be complementary. Inosine (I) has also been considered a universal base in the art and is considered to be complementary to any A, C, U, or T.

[0120] 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 in which the amino acid substitution is made. Variants can be prepared according to methods for changing polypeptide sequences known to those of skill in the art, and such methods are reviewed in the literature, e.g., Molecular Cloning: A Laboratory Manual, J. Sambrook et al., eds., 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2012, or Current Protocols in Molecular Biology, F.M. Ausubel et al., eds., John Wiley & Sons, Inc., New York. Conservative substitutions of amino acids include substitutions made between 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.

[0121] As used herein, the term "antibody" generally refers to a polypeptide that includes at least one immunoglobulin variable domain or at least one antigenic determinant, e.g., a paratope that specifically binds to an antigen. In some cases, the antibody is a full-length antibody. In some cases, the antibody is a chimeric antibody. In some cases, the antibody is a humanized antibody. However, in some cases, the antibody is a Fab fragment, an F(ab’)2 fragment, an Fv fragment, or a scFv fragment. In some cases, the antibody is a nanobody derived from a camelid antibody or a nanobody derived from a shark antibody. In some cases, the antibody is a diabody. In some cases, the antibody includes a framework having human germline sequences. In other cases, the antibody includes 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 cases, the antibody includes a heavy chain (H) variable region (abbreviated as VH herein) and / or a light chain (L) variable region (abbreviated as VL herein). In some cases, the antibody includes a constant domain, e.g., an Fc region. An immunoglobulin constant domain refers to a heavy chain constant domain or a light chain constant domain. The amino acid sequences and their functional variants of human IgG heavy chain constant domains and light chain constant domains are known. With respect to the heavy chain, in some cases, the heavy chain of the antibody disclosed herein can be an alpha (α), delta (δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In some cases, the heavy chain of the antibody disclosed herein can include a human alpha (α), delta (δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In certain cases, the antibody disclosed herein includes human gamma1 CH1, CH2, and / or CH3 domains. In some cases, the amino acid sequence of the VH domain includes the amino acid sequence of a human gamma (γ) heavy chain constant region known in the art. Non-limiting examples of human constant region sequences have been discussed in the art; see, e.g., U.S. Patent No., 5,693,780 and Kabat E A et al., (1991) supra.In some cases, the VH domain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or at least 99% identical to any of the variable chain constant regions provided herein. In some cases, the antibody is modified, for example, via glycosylation, phosphorylation, sumoylation and / or methylation. In some cases, the antibody is a glycosylated antibody conjugated to one or more sugar or carbohydrate molecules. In some cases, the one or more sugar or carbohydrate molecules are conjugated to the antibody via N-glycosylation, O-glycosylation, C-glycosylation, glypiation (GPI anchor attachment) and / or phosphoglycosylation. In some cases, the one or more sugar or carbohydrate molecules are monosaccharides, disaccharides, oligosaccharides or glycans. In some cases, the one or more sugar or carbohydrate molecules are branched oligosaccharides or branched glycans. In some cases, the one or more sugar or carbohydrate molecules comprise mannose units, glucose units, N-acetylglucosamine units, N-acetylgalactosamine units, galactose units, fucose units or lipid phosphate units. In some cases, the antibody is a construct comprising a polypeptide comprising one or more antigen-binding fragments of the present disclosure linked to a linker polypeptide or an immunoglobulin constant domain. The linker polypeptide comprises two or more amino acid residues linked by peptide bonds and is used to link one or more antigen-binding moieties. Examples of linker polypeptides have been reported (see, for example, Holliger, P. et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448; Poljak, R. J. et al. (1994) Structure 2: 1121-1123). Additionally, the antibody can be part of a larger immune adhesion molecule formed by covalent or non-covalent binding of the antibody or antibody portion to one or more other proteins or peptides.Examples of such immune adhesion molecules include the production of a tetrameric scFv molecule using a streptavidin core region (Kipriyanov, S.M. et al. (1995) Human Antibodies and Hybridomas 6: 93-101) and the production of divalent and biotinylated scFv molecules using cysteine residues, a marker peptide, and a C-terminal polyhistidine tag (Kipriyanov, S.M. et al. (1994) Mol. Immunol. 31: 1047-1058).

[0122] As used herein, the term "covalently bound" generally refers to the property that two or more molecules are linked to each other via at least one covalent bond. In some cases, two molecules can be covalently bound by a single bond that functions as an intermolecular linker, such as a disulfide bond or a disulfide bridge. However, in some cases, two or more molecules can be covalently bound to each other via a molecule that functions as a linker that binds two or more molecules via multiple covalent bonds. In some cases, the linker can be a cleavable linker. However, in some cases, the linker can be a non-cleavable linker.

[0123] As used herein, the term "specifically binds" generally refers to the ability of a molecule to bind to a binding partner with an affinity or binding activity to such an extent that the molecule can be used to distinguish the binding partner from appropriate controls in a binding assay or other binding context. With respect to an antibody, the term "specifically binds" is, as described herein, the degree of affinity or binding activity that can be used to distinguish a particular antigen from other antigens as compared to one or more appropriate reference antigens, and for example, refers to the ability of an antibody to bind to a specific antigen to such an extent that it can preferentially target Schwann cells via binding to the antigen. In some cases, the antibody has 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 K for binding to a target below M D When it has, the antibody specifically binds to the target.

[0124] 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) joined by peptide bonds, including modified amino acids (e.g., phosphorylation, glycosylation, glycation, etc.) and amino acid analogs. Exemplary polypeptides or proteins include gene products, natural proteins, homologs, paralogs, fragments and other equivalents, variants, and the above-mentioned analogs.

[0125] As used herein, the terms “subject,” “individual,” “mammal,” and “patient” are used interchangeably and generally refer to and mean that it may be a human as well as a non-human mammal (e.g., non-human primates, dogs, horses, cats, pigs, cows, ungulates, rabbits, etc.). In various instances, the subject can be a human (e.g., adult male, adult female, adolescent male, adolescent female, male child, female child) under the treatment of a physician or other healthcare provider as an outpatient in a hospital or in other clinical settings. In certain instances, the subject may not be under the treatment or prescription of a physician or other healthcare provider.

[0126] As used herein, the term “linker” generally refers to a molecule used to join two or more molecules. In certain instances, the linker can form a covalent bond with both molecules. Suitable linkers include, but are not limited to, straight-chain or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers.

[0127] If the terms "at least", "greater than", or "equal to or greater than" are present before the first numerical value of a series of two or more numerical values, the terms "at least", "greater than", or "equal to or greater than" apply to each numerical value of that series. For example, 1, 2, or 3 or greater is the same as 1 or greater, 2 or greater, or 3 or greater.

[0128] The terms "a", "an", and "the" include plural reference terms unless the context clearly indicates otherwise.

[0129] The term "about" means including a deviation of plus or minus 10 percent, particularly with respect to a given quantity.

[0130] Although various examples of the present disclosure are shown and described herein, it will be apparent to those skilled in the art that such examples are provided only by way of illustration. Numerous variations, modifications, and substitutions can be made by those skilled in the art without departing from the present disclosure. It should be understood that various alternative examples to the examples of the present disclosure described herein can be used.

Examples

[0131] The following is an explanation of various methods and materials used in the research, which is described for those skilled in the art to provide a complete disclosure and explanation of the preparation and use methods of the present disclosure. It is not intended to limit the scope considered by the inventors to be the disclosure, nor is it intended to represent that the following experiments were conducted and are all the experiments that can be conducted. Although efforts have been made to ensure the accuracy of the numerical values used (e.g., amounts, percentages, etc.), some experimental errors and deviations should be considered.

[0132] [Example 1] Evaluate Target Delivery In Vitro and In Vivo A complex of ML-LBP21 and GFP is generated.

[0133] The nucleic acid sequence of ML-LBP21 was synthesized and introduced into a bacterial expression system under the control of the lac promoter with a 6xHis tag at the C-terminus, in the presence and absence of green fluorescent protein (GFP), and then transformed into BL21 Escherichia coli. After induction with IPTG (0.1 mM, 16 h at 26 °C), the cells were centrifuged and harvested. The cells were lysed using sonication, and the soluble fraction was recovered. The cell supernatant was further purified on a HisTrap column and subsequently thoroughly washed and eluted using imidazole. The purified protein was then evaluated for purity by SDS-PAGE and analytical SEC. The binding affinity of ML-LBP21 to the leprosy receptor laminin 2 was also evaluated.

[0134] The delivery ability of the ML-LBP21 and GFP complex to Schwann cells was first tested by incubating the complex with primary Schwann cells or cell lines such as the mouse Schwann cell line MSC80, which is used as a neuropathological research system.

[0135] To determine whether the ML-LBP21-GFP complex or another detectable tag is targeted to Schwann cells, the ML-LBP21-GFP complex is co-incubated with primary cells or / and MSC80 cells. As a negative control, the cells are incubated with GFP only. As a positive control, primary cells or MSC80 cells are incubated with GFP using Lipofectamine reagent (Thermo Fisher). After incubation, the number of GFP-positive cells is detected by flow cytometry.

[0136] The total flux of GFP-positive cells is detected. The number of fluorescences detected in the group of primary cells or / and MSC80 cells incubated with the ML-LBP21-GFP complex is more than that incubated with GFP alone and is comparable to that incubated with GFP using Lipofectamine reagent. This demonstrates that ML-LBP21 promotes Schwann cell delivery.

[0137] To confirm that the uptake of the ML-LBP21-GFP complex into primary cells or / and MSC80 cells is actually mediated by ML-LBP21, a competitive uptake assay is performed. After primary cells or / and MSC80 cells are pre-incubated with the ML-LBP21 peptide at various concentrations, the cells are treated with the ML-LBP21-GFP complex, and simultaneously GFP alone is used as a control. The amount of the ML-LBP21-GFP complex detected in Schwann cells gradually decreases as the amount of the ML-LBP21 peptide increases. However, the low random uptake of GFP into cells is not affected by the pre-incubation with the ML-LBP21 peptide. This result indicates that the ML-LBP21-GFP complex enters Schwann cells through an ML-LBP21-mediated process.

[0138] Next, a complex of ML-LBP21 and GFP or other detectable tags is injected into wild-type mice. The mice are sacrificed at various time points, and the GFP / tag signal is detected in various tissues and organs and the isolated sciatic nerve. The GFP / tag signal is more concentrated in the isolated nerve. Further examination of the isolated nerve is performed by co-staining with cell markers that distinguish neurons (axons) from Schwann cells. The GFP / tag signal is concentrated in Schwann cells.

[0139] Similarly, ML-LBP21 is replaced with a targeting moiety that binds to gliomedin, and the above experiment is adopted to test a delivery system targeting gliomedin.

[0140] [Example 2] Evaluate the Targeted Delivery of siRNA Molecules In Vitro and In Vivo Complexes of siRNA targeting a gene (e.g., a housekeeping gene) and a targeting moiety (e.g., a ligand, a glycan ligand, ML-LBP21, an anti-gliomedin antibody) are generated. Double-stranded RNA (dsRNA) is conjugated to the targeting moiety (e.g., a ligand, a glycan ligand, ML-LBP21, an anti-gliomedin antibody).

[0141] To determine whether the ML-LBP21-siRNA complex is successfully delivered to Schwann cells, the ML-LBP21-siRNA complex is brought into contact with primary Schwann cells or / and MSC80 cells. As a negative control, primary cells or / and MSC80 cells are brought into contact with naked siRNA molecules. As a positive control, primary cells and / or MSC80 cells are incubated with GFP using the RNAiMax transfection reagent (Thermo Fisher). After incubation, cellular RNA is recovered and quantified by qPCR. The mRNA amount of the target gene in cells incubated with the ML-LBP21-siRNA complex is lower than that incubated with siRNA alone and is comparable to that incubated with siRNA using the RNAiMax transfection reagent.

[0142] Alternatively, the cells are genetically modified to express the target of the siRNA labeled with Ruby3. After incubation, the Ruby3 signal is reduced compared to that with siRNA alone and is equivalent to that of siRNA using the RNAiMax transfection reagent.

[0143] The above experiments demonstrate that ML-LBP21 promotes the delivery of siRNA to Schwann cells.

[0144] [Example 3] Evaluate the internalization of the targeting moiety in Schwann cells in vitro To identify antibodies that specifically bind to and internalize into Schwann cells, several antibodies that recognize extracellular epitopes of Schwann cell-expressed proteins were tested: mouse anti-myelin-associated glycoprotein (MAG) antibody mAb513 (MAB1567, Merck), rabbit anti-gliomedin antibody (raised against the olfactory factor domain of rat gliomedin), mouse anti-gliomedin antibody (mAb94, raised against residues 273-287 of the rat gliomedin protein, or raised against the binding residues 273-287 of the rat gliomedin protein (CVIPNDDTLVGRA)), and mouse anti-nectin-like protein 4 (Necl4, also known as Cadm4) antibody mAb244 / 5 (NeuroMAB). These antibodies were first labeled with a cy3-conjugated anti-mouse antibody (red). For rabbit-derived antibodies, Jackson ImmunoResearch Inc.'s Cy(trademark)3 AffiniPure Donkey Anti-Rabbit IgG(H+L) antibody (711-165-152) was used. For mouse-derived antibodies, Jackson ImmunoResearch Inc.'s Cy(trademark)3 AffiniPure Donkey Anti-Mouse IgG(H+L) antibody (715-165-151) was used. Next, live rat Schwann cell cultures were incubated with these antibodies for 30 minutes, followed by washing and further incubation in growth medium in an incubator for 48 hours. Thereafter, the cell cultures were fixed and stained with an anti-mouse 488 antibody (green) without performing cell permeabilization. Thus, yellow labeling indicates antibody molecules confirmed outside the cells, and red labeling indicates molecules confirmed inside the cells.

[0145] As shown in FIGS. 1A and 1B, the anti-Necl4 antibody (mAb244 / 5) and the anti-gliomedin antibody (mAb94) were able to bind to and effectively internalize into Schwann cells. However, FIG. 1C shows that the anti-MAG antibody was not internalized into Schwann cells.

[0146] The anti-gliomedin antibody (mAb94) was further investigated for its internalization ability. Rat Schwann cells were cultured on PLL-coated coverslips and incubated with the anti-gliomedin antibody (mAb94) pre-incubated with a cy3-conjugated anti-mouse antibody (red) for 30 minutes at 37°C. The cells were then washed and incubated in growth medium for the times shown in Figure 1D (i.e., 24 hours, 48 hours, or 72 hours). Thereafter, the cells were fixed. The fixed cells were labeled with an anti-mouse 488 antibody in the presence or absence of 0.1% Triton X100 in the antibody solution. Since Triton is a surfactant that permeates the cell membrane, the cell membrane remained intact without Triton, and only the anti-gliomedin antibody (mAb94) molecules confirmed outside the cells were co-labeled with the 488 antibody (green) along with the red label, and the internalized Ab molecules were only labeled red. In other words, extracellular and intracellular molecules were distinguished in the experiment without cell permeabilization treatment, but the entire antibody population was labeled in the experiment with cell permeabilization treatment. As shown in Figure 1D, after 24 hours, approximately half of the molecules remained co-labeled with 488 without permeabilization treatment, while half of the molecules were confirmed outside the cells. After 72 hours, a significant portion of the antibody population was internalized and thus not co-labeled with 488 without cell permeabilization treatment. In other words, co-localization of the green and red signals was confirmed only when Triton X was present and not when Triton X was absent, suggesting that the antibody was internalized into the cells (e.g., after 72 hours).

[0147] Next, the intracellular localization of the anti-gliomedin antibody (mAb94) internalized after 72 hours was investigated. As shown in Figure 1E, partial co-localization of the anti-gliomedin antibody (mAb94) with the lysosomal marker Lamp1 indicates that the anti-gliomedin antibody (mAb94) entered the lysosomes.

[0148] Furthermore, to test the internalization of the targeting moiety without the need for pre-incubation with the labeled secondary antibody, Cyanine5 NHS was conjugated and labeled to the anti-gliomedin antibody (mAb94) and control mouse IgG antibody. Antibody samples in PBS were mixed with a 10-fold solution of NaHCO3 (1M pH; 8.3) and Cyanine5 NHS (Lumiprobe cat# 43020) prepared as a stock solution (2.66 mg / ml DMSO) such that the molar ratio of the fluorophore to the antibody was 7:1. Tubes containing the reaction mixture were stored in the dark and incubated for 1 hour at room temperature with continuous mixing. The labeling reaction was stopped after 1 hour by adding 5 ml of 1M Tris-HCl pH 7.1 and dialysis was performed against PBS buffer.

[0149] Rat Schwann cells were incubated with the covalently labeled anti-gliomedin antibody (mAb94) and control IgG antibody (red) at 37°C for 30 minutes, followed by incubation in growth medium for 24 hours. The cells were fixed and stained with anti-mouse 488 without permeabilization to label extracellular antibody molecules. As shown in Figure 1F, the Cy3-labeled mAb94 antibody did not co-localize with the 488 signal, suggesting internalization into Schwann cells.

[0150] [Example 4] Evaluation of the specificity of the anti-gliomedin antibody (mAb94) An incubation setting similar to that disclosed in Example 3 was applied to primary cultures containing sensory neurons and Schwann cells prepared from dorsal root ganglia (DRGs) isolated from wild-type ("WT") or gliomedin-deficient mouse embryos ("KO"). Anti-gliomedin antibody (mAb94) was added to the cell cultures, and the cultures were further incubated. Anti-mouse 488 antibody (green) was applied without membrane permeabilization, followed by a second fixation and labeling with either a Schwann cell marker antibody ("MAG") or a neuronal marker Ab ("neurofilament"). As shown in FIGS. 2A and 2B, the anti-gliomedin antibody (mAb94) specifically internalized into Schwann cells in the mixed DRG neuron preparation. The signal from the anti-gliomedin antibody (mAb94) was not detected in receptor-deficient DRG cultures.

[0151] Cell type specificity was further confirmed. The DRG myelination cultures isolated from E13 mouse embryos contained three cell types: Schwann cells, sensory DRG neurons, and fibroblasts. Anti-gliomedin antibody (mAb94) antibody (green) was applied to live DRG cultures at 37°C for 30 minutes. Thereafter, the cultures were fixed and stained with marker antibodies (red), and neurons (neurofilament "NFH"), fibroblasts (vimentin "Vim"), and Schwann cells ("Cadm4")) were distinguished. The signal from the mAb94 antibody co-localized only with the Schwann cell marker antibody and was not detected at all in gliomedin-deficient cultures, suggesting that this antibody recognizes only gliomedin and does not cross-react with other proteins (see FIG. 2C).

[0152] Furthermore, the anti-gliomedin antibody (mAb94) recognized gliomedin from different species. COS7 cells were transfected with rat or human gliomedin. The cells were bound to the extracellular domain of NF186 (NF186FC, red). Thereafter, the cells were washed, fixed, and labeled with the anti-gliomedin antibody (mAb94). The mAb94 antibody bound to both rat and human gliomedin.

[0153] [Example 5] Evaluate the internalization of the antiglialmedin antibody (mAb94) into Schwann cells in vivo To test the targeting of the antiglialmedin antibody (mAb94) to Schwann cells in vivo, mice were injected via the tail vein with 150 μg of 650-mAb94 antibody (mice #1-3 and #4-6), control 650-IgG (#7-9 and #10-12), or nothing (mice #13-15). Mice were sacrificed 24 or 48 hours after injection. The sciatic nerves were harvested, fixed, and spread (fragmented) at single-fiber imaging resolution. The fragmented samples were labeled with an anti-caspr antibody (green), labeling the Ranvier nodes of myelinated nerve fibers. In the nerves of mice injected with 650mAb94, the antiglialmedin antibody (mAb94) (red) was strongly detected in clusters along the entire Ranvier nodes and myelin units (see mice #1-3 at 24 hours in Fig. 4A and mice #4-6 at 48 hours in Fig. 4B). The data indicate that the mAb94 antibody entered the nerve and bound to glialmedin on Schwann cells. In contrast, no significant signal was detected in the nerves of control-injected mice (see mice #7-9 at 24 hours in Fig. 4A, mice #10-12 at 48 hours in Fig. 4B) or non-injected mice (e.g., see mouse #13 in Fig. 4C). It was revealed that only the antiglialmedin antibody (mAb94) bound to myelinated nerves along the Ranvier nodes and Schwann cell nodes. However, the control IgG antibody showed no specific signal and had minimal staining similar to non-injected mice.

[0154] Therefore, these data suggest that the antiglialmedin antibody (mAb94) can cross the blood-nerve barrier and reach the nerve and be internalized into Schwann cells.

[0155] Furthermore, the distribution of the antiglialmedin antibody (mAb94) in various organs was evaluated using a fluorescence microscope.

[0156] Samples of various organs (n = 75) were collected from 15 mice after fluorescent labeling, fixed with 4% paraformaldehyde (PFA), and kept in the fixative for 48 hours for further fixation. The tissues were excised, placed in embedding cassettes, and processed for paraffin embedding using the protocol shown below. Four cassettes were prepared per animal.

[0157] The tissue processor was modified to a very short protocol to retain the fluorescent signal. 1. Formalin, 10 minutes at 37°C; 2. Water treatment, 2 minutes; 3. 70% ethanol, 5 minutes at 45°C; 4. 80% ethanol, 5 minutes at 45°C; 5. 95% ethanol, 5 minutes at 45°C; 6. 100% ethanol, 5 minutes at 45°C; 7. 100% ethanol, 5 minutes at 45°C; 8. 100% ethanol, 5 minutes at 45°C; 9. Xylene, 3 minutes at 45°C; 10. Xylene, 3 minutes at 45°C; 11. Xylene, 5 minutes at 45°C; 12. Paraffin, 5 minutes at 65°C; 13. Paraffin, 5 minutes at 65°C; 14. Paraffin, 10 minutes at 65°C.

[0158] Paraffin sections (4 μm thick) were cut out and placed on glass slides. The sections were evaluated for the fluorescent marker anti-gliomedin antibody (mAb94) using a fluorescence microscope.

[0159] The photographs shown in Figures 4D and 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 an objective magnification of X40 was scored using a semi-quantitative system: Grade 0: No detection of positively labeled cells; 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: More than 150 labeled cells were detected. Table 1 summarizes the specific localization and distribution of the detected antibody.

[0160]

Table 1

[0161] Generally, a distinct Cy5 signal was mainly confirmed in parts of the organs of the first and second groups. In the peripheral nerves, a distinct signal was confirmed in the nerve fibers, which diffused and spread along the nerve fibers. In the liver, a strong signal was only confirmed in the sinusoids, but this reaction was also confirmed in the negative control animals and untreated animals. In the spleen, a signal was also confirmed in the red pulp, but further examination revealed autofluorescence by red blood cells. No signal was confirmed in the kidneys and pancreas.

[0162] Most of the nerves in the first group (anti-gliomedin antibody (mAb94)) were signal positive (5 / 6), and a stronger signal was recorded at 48 hours compared to 24 hours after injection. In the second group (isotype control), one animal showed a very weak signal, which was probably due to the incomplete blocking process of the isotype Ab in the animal. In the third group (untreated), the animals were confirmed to be negative.

[0163] In summary, a fluorescently labeled anti-gliomedin antibody (mAb94) was intravenously injected into 12 animals. A prominent and reliable signal was only confirmed in the peripheral nerves. The signal at 48 hours after injection was stronger compared to the signal at 24 hours.

[0164] [Example 6] Evaluate the internalization of anti-laminin alpha 2 antibody into Schwann cells Antibodies that bind to laminin alpha-2 are further examined for their binding and internalization abilities into Schwann cells.

[0165] The antibody candidates include, but are not exclusive of other possibilities, antibody ABIN7439129 targeting amino acids 2901-3106 of laminin alpha-2, an antibody against laminin α2 G5 (2D4) from Abnova (Taipei, Taiwan), or a laminin alpha-2 monoclonal antibody (Thermo Fisher, #CL3450).

[0166] To assess the potential of the tested anti-laminin alpha-2 antibodies (e.g., Invitrogen CL3450) to bind to Schwann cells, rat sciatic nerve sections are used. The sciatic nerve is excised and fixed in 4% PFA for 20 minutes at room temperature (RT). The nerve is washed with PBS, spread (fragmented) on a slide glass, and individual fibers are isolated. The fragmented preparation is dried for 2 hours and stored at -20°C until use. For immunofluorescent labeling, the slides are incubated in a blocking solution (PBS, 5% normal goat serum, 0.5% TritonX100) for 1 hour at room temperature. The slides are then washed with PBS and incubated at 4°C overnight with the primary antibody: the tested anti-laminin alpha 2 antibody together with an anti-caspr antibody, labeling the myelinated axons of the Ranvier nodes. The primary antibody is diluted in an antibody solution (PBS, 5% normal goat serum, 0.1% Tritonx100). Subsequently, the slides are washed with PBS and incubated with the secondary antibody diluted in the antibody solution for 45 minutes at room temperature. The slides are then washed with PBS and mounted, for example, with fluormount-G (Invitrogen). The signal from the tested anti-laminin alpha 2 antibody is analyzed in relation to structures such as the Ranvier nodes and the basement membrane covering the myelinated Schwann cells.

[0167] The in vitro internalization ability of the tested anti-laminin alpha 2 antibody is also examined. Neurons (including Schwann cells) are freshly isolated from the sciatic nerve of mice, cultured on cover glasses coated with PLL, and incubated with the pre-labeled anti-laminin alpha 2 antibody. The neurons are then washed and incubated in growth medium for various times (e.g., 24 hours, 48 hours, or 72 hours). Thereafter, the neurons are fixed and labeled with a secondary antibody that recognizes the tested anti-laminin 2 antibody in the presence or absence of 0.1% Triton X-100 under various permeabilization conditions. The percentage of the tested anti-laminin alpha 2 antibody not co-labeled with the secondary antibody at different incubation periods without permeabilization is compared.

[0168] Furthermore, the cell type specificity of the tested anti-laminin 2 antibody is tested. A similar incubation setup is applied to primary cultures containing sensory neurons, fibroblasts, and Schwann cells that may be generated from DRGs isolated from either wild-type or laminin 2-deficient mouse embryos. Cell type markers such as neurons (neuron filaments), fibroblasts (vimentin), and Schwann cells (MAG) are used to distinguish different cell types.

[0169] Furthermore, the in vivo internalization ability of the tested anti-laminin alpha 2 antibody is examined. Mice are divided into groups and injected with the labeled anti-laminin alpha 2 antibody, control labeled IgG, or nothing. The mice are sacrificed 24 hours or 48 hours after injection. The sciatic nerve is harvested, fixed, and spread (fragmented) with single fiber image resolution. The fragmented samples are labeled with an anti-caspr antibody that labels the nodes of Ranvier of myelinated nerve fibers. The signal from the labeled anti-laminin 2 antibody is detected.

[0170] [Example 7] Evaluate the internalization of anti-Cadm4 / Necl4 antibody into Schwann cells The antibody that binds to Cadm4 / Necl4 is further examined for its ability to internalize into Schwann cells. Antibody mAb244 / 5 (NeuroMAB), which showed promising internalization results in Figure 1A, is further used. Other antibody candidates may include, but are not exclusive to, other possibilities such as the Necl4-Fc antibody of Eshed et al., Neuron, Volume 47, Issue 2, Pages 215 - 229, anti-SynCAM4 antibody (Biolegand, #833302) or IGSF4C / SynCAM4 antibody (rndsystems, #MAB41642). Schwann cells are cultured on PLL-coated cover glasses and incubated with pre-labeled anti-Cadm4 / Necl4 antibody. The cells are then washed and incubated in growth medium for different times (e.g., 24 hours, 48 hours or 72 hours). Thereafter, the cells are fixed and labeled with a secondary antibody that recognizes the tested anti-Cadm4 / Necl4 antibody in the presence or absence of 0.1% Triton X-100 under various permeabilization conditions. The percentage of the tested anti-Cadm4 / Necl4 antibody that is not co-labeled with the secondary antibody at different incubation periods without permeabilization is compared.

[0171] Furthermore, the cell type specificity of the tested anti-Cadm4 / Necl4 antibody is tested. A similar incubation setup is applied to primary cultures containing sensory neurons, fibroblasts and Schwann cells that may be generated from DRGs isolated from wild-type or Cadm4 / Necl4-deficient mouse embryos. Cell type markers such as neurons (neuron filaments), fibroblasts (vimentin) and Schwann cells (MAG) are used to distinguish different cell types.

[0172] Furthermore, the in vivo internalization ability of the tested anti-Cadm4 / Necl4 antibodies is further investigated. Mice are divided into groups and injected with labeled anti-Cadm4 / Necl4 antibodies, control labeled IgG, or nothing. The mice are sacrificed 24 or 48 hours after injection. The sciatic nerve is harvested, fixed, and spread (sectioned) at single fiber resolution. The sectioned samples are labeled with an anti-caspr antibody that labels the nodes of Ranvier of myelinated nerve fibers. Signals from the labeled anti-Cadm4 / Necl4 antibodies are detected.

[0173] It should be understood that the various aspects of the present disclosure can be evaluated individually, collectively, or in combination with each other. The various aspects of the present disclosure described herein can be applied to any of the specific uses disclosed herein. The compositions of substances disclosed in the composition items of the present disclosure can be utilized in the method items of methods including the methods of use and manufacture disclosed herein, and vice versa.

[0174] Although preferred examples of the present disclosure are shown and described herein, it will be apparent to those skilled in the art that such examples are provided by way of illustration only. The present disclosure is not intended to be limited by the specific examples provided herein. Although the present disclosure has been described with reference to the foregoing specification, the description and illustration of the examples herein are not intended to be construed in a limiting sense. Numerous variations, modifications, and alternatives will be understood by those skilled in the art without departing from the present disclosure. Furthermore, it should be understood that all aspects of the present disclosure are not limited to the specific depictions, configurations, or relative proportions described herein, which depend on various conditions and elements of variation. It should be understood that various alternatives to the examples of the present disclosure described herein can be employed in the practice of the present disclosure. Accordingly, the present disclosure is also intended to cover such alternatives, modifications, variations, or equivalents. The following claims define the scope of the present disclosure, and it is intended that methods and structures within these claims and their equivalents be covered thereby.

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