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WO2025150052A1 - Administration ciblée de conjugués de médicament à des cellules de schwann et méthodes de traitement dans des maladies associées à des cellules de schwann - Google Patents

Administration ciblée de conjugués de médicament à des cellules de schwann et méthodes de traitement dans des maladies associées à des cellules de schwann

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Publication number
WO2025150052A1
WO2025150052A1 PCT/IL2025/050035 IL2025050035W WO2025150052A1 WO 2025150052 A1 WO2025150052 A1 WO 2025150052A1 IL 2025050035 W IL2025050035 W IL 2025050035W WO 2025150052 A1 WO2025150052 A1 WO 2025150052A1
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cdr
antibody
instances
molecule
drug
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Inventor
Oren Bogin
Hila EPSTEIN BARASH
Barak Azmon
Yair Alster
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Potentia Ltd
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Potentia Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/68031—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being an auristatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6807—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug or compound being a sugar, nucleoside, nucleotide, nucleic acid, e.g. RNA antisense
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity

Definitions

  • Schwann cells also called neurilemma cells, produce the myelin sheath around neuronal axons in the peripheral nervous system. They play a key role in the pathology of various inflammatory, metabolic, hereditary neuropathies and cancer (see, e.g., Lehmann and Hoke, CNS Neurol Disord Drug Targets. 2010 Dec, 9(6): 801-806; Kamil K, et al. Front. Neurol. 2019,10:87; Kresak, J.L, and Walsh, M., J Pediatr Genet. 2016, 5: 98, which are incorporated by reference herein in its entirety).
  • peripheral neuropathies are associated with abnormal protein or gene expression in Schwann cells, and suppression or regulation of such protein or gene expression in Schwann cells has been contemplated to treat those peripheral neuropathies.
  • the drug delivery methods in the current practice such as deliveries via nanoparticle and viral delivery, such as Adeno-associated virus (AAV) possess disadvantages including delivery mode, nonspecific targeting, reduced targeting efficiency, immunogenicity, generation of anti-drug antibodies (AD As) or safety concerns.
  • AAV Adeno-associated virus
  • the present disclosure in one aspect, discloses a drug conjugate comprising a targeting moiety conjugated to a drug molecule, wherein the targeting moiety binds to a cell adhesion moiety and/or receptor expressed on a Schwann cell to mediate targeted delivery of the drug conjugate to the Schwann cell, and wherein the drug molecule modifies expression or activity of a disease-associated molecule, and/or confers a cytotoxic effect, in the Schwann cell.
  • a drug conjugate comprising a targeting moiety conjugated to a drug molecule, wherein the targeting moiety binds to a cell adhesion moiety and/or receptor expressed on a Schwann cell to mediate targeted delivery of the drug conjugate to the Schwann cell, and wherein the drug molecule modifies expression or activity of a disease- associated molecule, and/or confers a cytotoxic effect, in the Schwann cell.
  • the cell adhesion moiety and/or receptor is selected from Nectin-like protein (Neel) e.g. Necll, Necl3, Necl2 and Necl4, SynCAM e.g. SynCAM4, Cadm e.g. Cadml- Cadm3, Neurofascinl55 (NF155), TAG1, myelin-associated glycoprotein (MAG), and neuronal cell adhesion molecule (NrCAM).
  • Nectin-like protein Nectin-like protein
  • Necl3, Necl2 and Necl4 SynCAM e.g. SynCAM4, Cadm e.g. Cadml- Cadm3, Neurofascinl55 (NF155), TAG1, myelin-associated glycoprotein (MAG), and neuronal cell adhesion molecule (NrCAM).
  • the drug conjugate as described in any of the above, wherein the targeting moiety is an antibody or antigen binding fragment thereof or a ligand molecule.
  • the targeting moiety is selected from anti-human Gliomedin Antibody, anti- Cadm4 mAb244/5 (NeuroMAB), Necl4-Fc antibody, anti-SynCAM4 Antibody, IGSF4C/SynCAM4 Antibody, neurofascin- 186 (NF 186) or a portion or derivative thereof, glycolipid PGL-1 or a portion or derivative thereof, trisaccharide of PGL-1 or a portion or derivative thereof, ML-LBP21 (histone-like protein/Hlp) or a portion or derivative thereof.
  • the targeting moiety is selected from anti-human Gliomedin Antibody, anti- Cadm4 mAb244/5 (NeuroMAB), Necl4-Fc antibody, anti-SynCAM4 Antibody, IGSF4C/SynCAM4 Antibody, neurofascin- 186 (NF 186) or a portion or derivative thereof, glycolipid PGL-1 or a portion or derivative thereof, trisacchari
  • the antibody or antigen binding fragment thereof comprises IgG (Immunoglobulin G), including IgGl, IgG2a IgG2b, IgG3 and IgG4, IgA (Immunoglobulin A), including IgAl and IgA2, IgM (Immunoglobulin M), IgE (Immunoglobulin E), IgD (Immunoglobulin D), fragment antigen-binding (Fab fragment), fragment crystallizable region (Fc region), monovalent Fab’, divalent Fab2, single-chain variable fragment (scFv), diabody, minibody, nanobody, single-domain antibody (sdAb), (scFv)2, Fab, Fab', F(ab')2, Fv, dAb, Fd fragments, diabodies, F(ab')3, disulfide linked Fv, sdAb (IgG (Immunoglobulin G), including IgGl, IgG2a Ig
  • the cell adhesion moiety or receptor as disclosed herein is selected from a group consisting of Gliomedin, a leprosy receptor (e.g., Laminin alpha 2-G4-5 (LNa2G), P0 protein and alpha-Dystroglycan (alpha-DG)), TAM (Tyro3, Axl, Mer) receptor, cell adhesion molecule (Cadm), or any Schwann cell specific receptor.
  • the cell adhesion moiety or receptor is Gliomedin.
  • the cell adhesion moiety or receptor is Cadm.
  • the cell adhesion moiety or receptor is a leprosy e.g., Laminin alpha 2-G4-5 (LNa2G), alpha-Dystroglycan (alpha-DG) and Myelin Protein zero (P0 protein).
  • the cell adhesion moiety or receptor is Tyro3, Axl, Mer (TAM) receptor.
  • the cell adhesion moiety and/or receptor is selected from Nectin- like protein (Neel) e.g. Necll, Necl3, Necl2 and Necl4, SynCAM e.g. SynCAM4, Cadm e.g.
  • the targeting moiety is an antibody or antigen, a binding fragment thereof or a ligand molecule.
  • the antibody is a fully human antibody, and is recombinantly produced.
  • the antibody or antigen binding fragment thereof may comprise IgG (Immunoglobulin G), including IgGl, IgG2a IgG2b, IgG3 and IgG4, IgA (Immunoglobulin A), including IgAl and IgA2, IgM (Immunoglobulin M), IgE (Immunoglobulin E), IgD (Immunoglobulin D), fragment antigenbinding (Fab fragment), fragment crystallizable region (Fc region), monovalent Fab’, divalent Fab2, single-chain variable fragment (scFv), diabody, minibody, nanobody, single-domain antibody (sdAb), (scFv)2, Fab, Fab', F(ab')2, Fv, dAb, Fd fragments, diabodies, F(ab')3, disulfide linked Fv, sdAb (VHH or nanobody), CDR (Complementarity-determining region), di-scFv, bi
  • the targeting moiety is any antigen-binding variant that comprises a CDR sequence having at least 90% sequence identity, such as at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% to a sequence selected from SEQ ID NO: 3-22, and 30 or any fragment or functional variant thereof.
  • the drug molecule comprises a peptide or a small molecule, such as an oligonucleotide and/or toxin.
  • the drug molecule comprises toxins commonly or conventionally used, as an example, Auristatin derivative such as Monomethyl auristatin E (MMAE) and Monomethylauristatin F (MMAF).
  • Auristatin derivative such as Monomethyl auristatin E (MMAE) and Monomethylauristatin F (MMAF).
  • toxins, cell death inducing molecules and cytotoxic agents are herein as drug molecules within the drug conjugate for treatment of a nerve tumor disease such as schwannomatosis, schwannomas and neurofibromatosis type 1 (NF1).
  • NF1 neurofibromatosis type 1
  • the Drug molecule to the Targeting Moiety Ratio is about 1 to 10.
  • the Drug-Targeting Moiety Ratio (DTMR), as measured according to Matsuda, Y., Mendelsohn, BA., Chem Pharm Bull (Tokyo). 2021 ;69, 976 is between 2 to 8. In some instances, the DTMR can potentially range from 0 to 8. In some instances, an average DTMR is between 3 to 4.
  • DTMR DTMR
  • the specific DTMR may vary depending on several factors, including: [0077] - The type of the targeting moiety (e.g. antibody): Different targeting moieties (e.g. antibodies) have different structures and properties that can affect the amount of drug molecules that can be attached.
  • the desired therapeutic effect is to find a balance between delivering sufficient drug to kill cancer cells or to modify expression or activity of a disease associated molecule, while minimizing side effects.
  • the drug conjugate of the present invention is aimed to achieve the maximal therapeutic efficacy with minimum drug dosage.
  • homolog and “homologous sequence” are often used to describe genetic sequences that share a common ancestry and exhibit a certain degree of identity or similarity.
  • the term “homolog” refers to a polynucleotide or polypeptide sequence that shares a common evolutionary origin with another sequence (e.g. gene or protein or any fragment thereof). Homologs can be found within the same species (paralogs) or in different species (orthologs). They typically perform similar functions but may have diverged over time due to evolutionary pressures.
  • homologous sequence refers to a DNA, RNA, or protein (polypeptide) sequence that is similar to another sequence.
  • similarity and identity additionally refer to local homology, identifying domains that are homologous or similar (in nucleotide and/or amino acid sequence). It is acknowledged that bioinformatics tools such as BLAST, S SEARCH, FAS TA, and HMMER calculate local sequence alignments which identify the most similar region between two sequences. For domains that are found in different sequence contexts in different proteins, the alignment should be limited to the homologous domain, since the domain homology is providing the sequence similarity captured in the score.
  • sequence motif which is a nucleotide or amino-acid sequence pattern that is widespread and has, or is conjectured to have, a biological significance.
  • Proteins may have a sequence motif and/or a structural motif, a motif formed by the three- dimensional arrangement of amino acids which may not be adjacent.
  • the amount of sequence identity shared by a target and a donor polynucleotide or polypeptide can vary and includes total lengths and/or regions having unit integral values in the ranges of about 1-20, 20-50, 50-100, 75-150, 100-250, 150-300, 200-400, 250- 500, 300-600, 350-750, 400-800, 450-900, 500-1000, 600-1250, 700- 1500, 800-1750, 900-2000, l-2.5k, 1.5- 3 k, 2-4 k, 2.5-5 k, 3-6 k, 3.5-7 k, 4-8 k, 5-10 k base pairs or amino acids, or up to and including the total length of the target site. These ranges include every integer within the range.
  • the structural similarity between a given sequence region and the corresponding region of homology can be of any degree of sequence identity that allows for homologous recombination or hybridization, binding or recognition to occur.
  • the amount of homology or sequence identity shared by two sequences can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, such that the sequences undergo homologous recombination, hybridization or recognition or binding.
  • the term "functional variant” or “functional variant of a nucleic acid or amino acid sequence” as used herein refers to a sequence or part or fragment of a sequence which retains the biological function of the full non-variant and hence has the activity of the expressed gene or polypeptide.
  • a functional variant also comprises a variant of a nucleic acid sequence of interest encoding a polypeptide which has sequence alterations that do not affect function of the resulting polypeptide, for example, in non-conserved residues or by degenerate, or redundant sequence encoding the same amino acids.
  • antibody humanization or “humanization” or “humanized antibody” which refers to a biotechnological process used to modify non- human antibodies, typically derived from animals like mice, to make them more compatible with the human immune system. This technique is crucial for developing therapeutic antibodies that are less likely to trigger immune responses, such as the production of anti-drug antibodies (AD As), which can neutralize the drug’s effects and cause adverse reactions.
  • AD As anti-drug antibodies
  • the process involves replacing the non-human constant and framework regions of the antibody with human sequences while preserving the antigen-binding complementarity-determining regions (CDRs), which maintain the antibody’s specificity.
  • the humanization process includes sequencing the non-human antibody and selecting a human framework with structural similarity and low immunogenic potential.
  • the CDRs are then grafted onto this framework, followed by computational and experimental optimization to enhance binding affinity, stability, and expression levels.
  • Advanced techniques such as deimmunization and structure-based design, further reduce immunogenicity and improve functionality.
  • Humanized antibodies have become essential in treating various diseases, including cancers, autoimmune disorders, and infectious diseases, as they combine high specificity and therapeutic efficacy with minimized risk of immune rejection. Humaniztion of antibodies are described for example in WO1991009967A1. Conjugates
  • peripheral nervous system PNS
  • oncology related disorders has focused on non-targeted therapeutics or surgery, but the treatment has been shown with poor clinical outcomes.
  • PNS disorders the PNS disorders that are directly or indirectly related to the dysfunction of the Schwann cells, specific or preferred targeting of drugs to the Schwann cell can increase the efficacy and outcome of the treatment.
  • the specificity of the manipulation and/or modification of Schwann cells is a main roadblock to advance Schwann cell therapies.
  • the target moiety that specifically directs cargoes or paylaoads to Schwann cells.
  • the cell adhesion moiety or receptor complex comprises an extracellular molecule that forms a complex with the cell adhesion moiety or receptor molecule (e.g., membrane bound cell adhesion moiety or receptor via transmembrane domain) directly or indirectly.
  • the targeting moiety binds to the extracellular molecule of the complex.
  • the targeting moiety binds to a cell adhesion molecule that mediates the interaction between myelinating Schwann cells and the axons they ensheath.
  • the cell adhesion moiety or receptor is expressed in the myelinated Schwann cells.
  • the cell adhesion moiety or receptor is highly expressed at the edge of myelin cells.
  • the cell adhesion moiety or receptor mediates the interaction of axon and glial cells or mediates axon-glial contact. In some instances, the cell adhesion moiety or receptor mediates the pathogen (or a portion thereof) uptake to the Schwan cells in certain disease conditions.
  • the targeting moiety binding to a cell adhesion moiety or receptor also includes a targeting moiety that binds to a portion of a cell adhesion moiety or receptor complex that are naturally present in healthy or pathological conditions of Schwann cells. [0105]
  • the targeting moiety is an antibody or antigen binding fragment thereof or a ligand molecule.
  • the antibody or antigen binding fragment thereof comprises IgG (Immunoglobulin G), including IgGl, IgG2a IgG2b, IgG3 and IgG4 isotypes, IgA (Immunoglobulin A), including IgAl and IgA2 isotypes, IgM (Immunoglobulin M), IgE (Immunoglobulin E), IgD (Immunoglobulin D), fragment antigen-binding (Fab fragment), fragment crystallizable region (Fc region), monovalent Fab’, divalent Fab2, singlechain variable fragment (scFv), diabody, minibody, nanobody, single-domain antibody (sdAb), (scFv)2, Fab, Fab', F(ab')2, Fv, dAb, Fd fragments, diabodies, F(ab')3, disulfide linked Fv, sdAb (VHH or nanobody), CDR (Complementarity
  • the targeting moiety disclosed herein binds to a portion of the C- terminal region of a gliomedin. In some instances, the targeting moiety disclosed herein binds to the coiled-coil domain of a gliomedin. In some instances, the targeting moiety disclosed herein binds to the interrupted collagen repeats domain of a gliomedin. In some instances, the targeting moiety disclosed herein binds to the olfactomedin domain of a gliomedin. In some instances, the targeting moiety disclosed herein binds to amino acid residues 276-287 of rat Gliomedin NP 852047.2 (CVIPNDDTLVGRA).
  • the targeting moiety disclosed herein binds to amino acid residues 365-460 of a Gliomedin.
  • the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof which binds to a Gliomedin.
  • the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof which binds to the extracellular region of a gliomedin.
  • the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof which binds to the C-terminal region of a gliomedin.
  • the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof which binds to a portion of the C-terminal region of a gliomedin.
  • the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof which binds to the coiled-coil domain of a gliomedin. In some instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof which binds to the interrupted collagen repeats domain of a gliomedin. In some instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof which binds to the olfactomedin domain of a gliomedin. In some instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof which binds to amino acid residues 276-287 of rat Gliomedin NP 852047.2 (CVIPNDDTLVGRA). In some instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof which binds to amino acid residues 365-460 of a gliomedin.
  • the targeting moiety disclosed herein is a commercially available antibody that binds to gliomedin.
  • the targeting moiety disclosed herein is anti-Gliomedin antibody (mAb94) disclosed in Eshed etal., Neuron, volume 47, issue 2, p215- 229, which is incorporated by reference in its entirety.
  • the targeting moiety disclosed herein is anti-GLDN antibody NovoPro #: 175453.
  • the targeting moiety disclosed herein is anti-GLDN antibody Thermo Fisher #:BS-11032R.
  • the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof which binds to a leprosy receptor. In specific instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof which binds to laminin 2. In specific instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof which binds to the C-terminal region of laminin 2. In some instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof which binds to the a2-subunit of laminin 2. In some instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof which binds to G domain of the a2-subunit of laminin 2.
  • the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof which binds to G5 domain of the a2-subunit of laminin 2. In some instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof which binds to the 01 -subunit of laminin 2. In some instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof which binds to the yl -subunit of laminin 2. In some instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof which binds amino acids 2901-3106 of lamin-2. In other instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof which binds to PO protein (myelin protein zero).
  • the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof which binds to an N-terminal extracellular immunoglobulin (Ig)-like domain of PO protein. In some instances, the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof which binds to the N-linked oligosaccharide in the extracellular domain of the leprosy receptor or any subunits thereof, and/or the addition of sulfate, acyl, and phosphate groups.
  • Ig extracellular immunoglobulin
  • the targeting moiety disclosed herein is a commercially available antibody that binds to laminin-2. In some instances, the targeting moiety disclosed herein is anti-laminin antibody ABIN7439129. In some instances, the targeting moiety disclosed herein is anti- laminin alpha-2 monoclonal antibody (Thermo Fisher, #CL3450).
  • the targeting moiety disclosed herein binds to a cell adhesion molecule (Cadm). In some instances, the targeting moiety disclosed herein binds to Cadml. In some instances, the targeting moiety disclosed herein binds to IgV-set domain of Cadml or a portion thereof. In some instances, the targeting moiety disclosed herein binds to IgCl-set domain of Cadml or a portion thereof. In some instances, the targeting moiety disclosed herein binds to Igl-set domain of Cadml or a portion thereof. In specific instances, the targeting moiety disclosed herein binds to Cadm2.
  • Cadm cell adhesion molecule
  • the targeting moiety disclosed herein binds to IgV-set domain of Cadm2 or a portion thereof. In some instances, the targeting moiety disclosed herein binds to IgCl-set domain of Cadm2 or a portion thereof. In some instances, the targeting moiety disclosed herein binds to Igl-set domain of Cadm2 or a portion thereof. In some instances, the targeting moiety disclosed herein binds to Cadm3. In some instances, the targeting moiety disclosed herein binds to IgV-set domain of Cadm3 or a portion thereof. In some instances, the targeting moiety disclosed herein binds to IgCl-set domain of Cadm3 or a portion thereof.
  • the targeting moiety disclosed herein an antibody or antigen binding fragment thereof which binds to a cell adhesion molecule (Cadm). In some instances, the targeting moiety disclosed herein an antibody or antigen binding fragment thereof which binds to Cadml. In some instances, the targeting moiety disclosed herein an antibody or antigen binding fragment thereof which binds to IgV-set domain of Cadml or a portion thereof. In some instances, the targeting moiety disclosed herein an antibody or antigen binding fragment thereof which binds to IgCl -set domain of Cadml or a portion thereof. In some instances, the targeting moiety disclosed herein an antibody or antigen binding fragment thereof which binds to Igl-set domain of Cadml or a portion thereof.
  • the targeting moiety disclosed herein an antibody or antigen binding fragment thereof which binds to Igl-set domain of Cadml or a portion thereof.
  • the targeting moiety disclosed herein an antibody or antigen binding fragment thereof which binds to IgV- set domain of Cadm3 or a portion thereof. In some instances, the targeting moiety disclosed herein an antibody or antigen binding fragment thereof which binds to IgCl-set domain of Cadm3 or a portion thereof. In some instances, the targeting moiety disclosed herein an antibody or antigen binding fragment thereof which binds to Igl-set domain of Cadm3 or a portion thereof. In some instances, the targeting moiety disclosed herein an antibody or antigen binding fragment thereof which an antibody or antigen binding fragment thereof which binds to Cadm4.
  • the targeting moiety disclosed herein an antibody or antigen binding fragment thereof which binds to IgV-set domain of Cadm4 or a portion thereof. In some instances, the targeting moiety disclosed herein an antibody or antigen binding fragment thereof which binds to IgCl -set domain of Cadm4 or a portion thereof. In some instances, the targeting moiety disclosed herein an antibody or antigen binding fragment thereof which binds to Igl-set domain of Cadm4 or a portion thereof.
  • the targeting moiety disclosed herein is a commercially available antibody that binds to Cadml. In some instances, the targeting moiety disclosed herein is a commercially available antibody that binds to Cadm2. In some instances, the targeting moiety disclosed herein is a commercially available antibody that binds to Cadm3. In some instances, the targeting moiety disclosed herein is a commercially available antibody that binds to Cadm4. In some instances, the targeting moiety disclosed herein is anti-Cadm4 mAb244/5 (NeuroMAB). In some instances, the targeting moiety disclosed herein is anti-human Gliomedin Antibody.
  • the targeting moiety disclosed herein is Necl4-Fc antibody from Eshed et al., Neuron, volume 47, issue 2, p215-229. In some instances, the targeting moiety disclosed herein is anti-SynCAM4 Antibody (Biolegand, #833302). In some instances, the targeting moiety disclosed herein is IGSF4C/SynCAM4 Antibody (rndsystems, #MAB41642).
  • the targeting moiety directs the conjugate to Schwann cells, and the conjugates are internalized upon the binding of the targeting moiety to the cell adhesion moiety or receptor, which is about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, or up to 90 days after contacting the Schwann cell.
  • the targeting moiety directs the conjugate to Schwann cells, and the conjugates are internalized upon the binding of the targeting moiety to the receptor, which is within about 24 hours, about 36 hours, about 48 hours, about 60 hours, or up to 90 days after contacting the Schwann cell.
  • the targeting moiety disclosed herein directs and internalizes the conjugate disclosed herein more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours after contacting the Schwann cell. In some instances, the targeting moiety disclosed herein directs and internalizes the conjugate disclosed herein less than 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, or 96 hours after contacting the Schwann cell.
  • the drug molecules disclosed herein are released after being internalized into the Schwann cells. In some instances, the drug molecules disclosed herein are released after the conjugates disclosed herein enter a lysosome of the Schwann cells. In some instances, the drug molecules disclosed herein are released to the cytoplasm of the Schwann cells. In some instances, the drug molecules disclosed herein are released to the nucleus of the Schwann cells.
  • the targeting moiety disclosed herein is an antibody or antigen binding fragment thereof.
  • the antibody or antigen binding fragment thereof comprises IgG (Immunoglobulin G), including IgGl, IgG2a IgG2b, IgG3 and IgG4, IgA (Immunoglobulin A), including IgAl and IgA2, IgM (Immunoglobulin M), IgE (Immunoglobulin E), IgD (Immunoglobulin D), fragment antigen-binding (Fab fragment), fragment crystallizable region (Fc region), monovalent Fab’, divalent Fab2, single-chain variable fragment (scFv), diabody, minibody, nanobody, single-domain antibody (sdAb), or camelid antibody or binding fragment thereof.
  • IgG Immunoglobulin G
  • IgG2a IgG2b IgG3 and IgG4
  • IgA Immunoglobulin A
  • IgM Immunoglob
  • the targeting moiety disclosed herein is a combination of one or more of the above-mentioned antibodies, antigen binding fragments thereof, or ligand molecules.
  • the targeting moiety disclosed herein comprises at least a portion of neurofascin-186 (NF186) or a derivative thereof. In some instances, the targeting moiety disclosed herein comprises one or more gliomedin-binding domain of NF 186.
  • the targeting moiety disclosed herein comprises at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, or 60% of full length NF 186 without significantly affecting the binding affinity to gliomedin. In some instances, the targeting moiety disclosed herein comprises no greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of full length NF 186 without significantly affecting the binding affinity to gliomedin. In some instances, the targeting moiety disclosed herein comprises a portion of NF 186 with one or more conservative amino acid substitutions without significantly affecting the binding affinity to gliomedin.
  • the targeting moiety disclosed herein comprises a portion of NF 186 with one or more substitution with an alpha, a beta, a gamma, or a delta amino acid without significantly affecting the binding affinity to gliomedin. In some instances, the targeting moiety disclosed herein comprises a portion of NF 186 with one or more substitution with a positively-charged, negatively-charged, or neutral amino acid without significantly affecting the binding affinity to gliomedin. In some instances, the targeting moiety disclosed herein comprises a portion of NF 186 with one or more substitution with a polar or non-polar amino acid without significantly affecting the binding affinity to gliomedin.
  • the targeting moiety disclosed herein comprises a portion of NF186 with one or more amino acid deletions without significantly affecting the binding affinity to gliomedin. In some instances, the targeting moiety disclosed herein comprises a portion of NF186 with one or more amino acid insertions without significantly affecting the binding affinity to gliomedin. In some instances, the targeting moiety disclosed herein comprises a portion of NF 186 and an amino acid sequence which is at least partly the reverse of the amino acid sequence of the corresponding portion of NF 186, without significantly affecting the binding affinity to gliomedin.
  • the targeting moiety disclosed herein comprises a portion of NF 186, and wherein at least one of the amino acids from the original portion of NF 186 is replaced by a stereoisomer (e.g., D-stereoisomer) of that amino acid, without significantly affecting the binding affinity to gliomedin.
  • the targeting moiety disclosed herein comprises a portion of NF186, and is extended at one or both ends thereof with one or more groups, such as D-amino acids, without significantly affecting the binding affinity to gliomedin.
  • the targeting moiety disclosed herein comprises NF 186 with a combination of one or more of the above-mentioned modifications without significantly affecting the binding affinity to gliomedin.
  • the targeting moiety disclosed herein comprises a recombinant NF186 peptide or a derivative thereof. In some instances, the targeting moiety disclosed herein comprises a gliomedin-binding domain of recombinant NF186. In some instances, the targeting moiety disclosed herein comprises at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, or 60% of full length recombinant NF186 without significantly affecting the binding affinity to gliomedin. In some instances, the targeting moiety disclosed herein comprises no greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of full length recombinant NF 186 without significantly affecting the binding affinity to gliomedin.
  • the targeting moiety disclosed herein comprises a portion of recombinant NF 186 with one or more conservative amino acid substitutions without significantly affecting the binding affinity to gliomedin. In some instances, the targeting moiety disclosed herein comprises a portion of recombinant NF 186 with one or more non-conservative amino acid substitutions without significantly affecting the binding affinity to gliomedin. In some instances, the targeting moiety disclosed herein comprises a portion of recombinant NF 186 with one or more substitution with a basic, an acidic, an aromatic, an aliphatic, an acid amide, a cyclic, or a sulfur-containing amino acid without significantly affecting the binding affinity to gliomedin.
  • the targeting moiety disclosed herein comprises a portion of recombinant NF 186 with one or more substitution with a standard amino acid or a non-standard amino acid without significantly affecting the binding affinity to gliomedin. In some instances, the targeting moiety disclosed herein comprises a portion of recombinant NF186 with one or more substitution with an alpha, a beta, a gamma, or a delta amino acid without significantly affecting the binding affinity to gliomedin. In some instances, the targeting moiety disclosed herein comprises a portion of recombinant NF 186 with one or more substitution with a positively-charged, negatively - charged, or neutral amino acid without significantly affecting the binding affinity to gliomedin.
  • the targeting moiety disclosed herein comprises a portion of recombinant NF186 with one or more substitution with a polar or non-polar amino acid without significantly affecting the binding affinity to gliomedin. In some instances, the targeting moiety disclosed herein comprises a portion of recombinant NF 186 with one or more amino acid deletions without significantly affecting the binding affinity to gliomedin. In some instances, the targeting moiety disclosed herein comprises a portion of recombinant NF186 with one or more amino acid insertions without significantly affecting the binding affinity to gliomedin.
  • the targeting moiety disclosed herein comprises a portion of recombinant NF 186 and an amino acid sequence which is at least partly the reverse of the amino acid sequence of the corresponding portion of recombinant NF 186 without significantly affecting the binding affinity to gliomedin.
  • the targeting moiety disclosed herein comprises a portion of recombinant NF 186, and wherein at least one of the amino acids from the original portion of recombinant NF 186 is replaced by a stereoisomer (e.g., D-stereoisomer) of that amino acid without significantly affecting the binding affinity to gliomedin.
  • the targeting moiety disclosed herein comprises a portion of recombinant NF 186, and is extended at one or both ends thereof with one or more groups, such as D-amino acids, without significantly affecting the binding affinity to gliomedin. In some instances, the targeting moiety disclosed herein comprises recombinant NF 186 with a combination of one or more of the above-mentioned modifications without significantly affecting the binding affinity to gliomedin.
  • the targeting moiety disclosed herein binds to G3 domain of the a2-subunit of laminin 2. In some instances, the targeting moiety disclosed herein binds to G4 domain of the a2-subunit of laminin 2. In some instances, the targeting moiety disclosed herein binds to G5 domain of the a2-subunit of laminin 2. In some instances, the targeting moiety disclosed herein binds to the 01 -subunit of laminin 2. In some instances, the targeting moiety disclosed herein binds to the yl -subunit of laminin 2.
  • the targeting moiety disclosed herein comprises at least a portion of glycolipid PGL-1 or a derivative thereof. In some instances, the targeting moiety disclosed herein comprises the unique trisaccharide of PGL-1 (3,6-di-O-methylglucose linked a-1— >4 to 2,3-di-O-methylrhamnose linked 0-1 — >2 to 3-O-methylrhamnose).
  • the targeting moiety disclosed herein comprises at least a portion of ML-LBP21 (also known histone-like protein/Hlp) or a derivative thereof. In some instances, the targeting moiety disclosed herein comprises a laminin-2-binding domain of ML-LBP21. In some instances, the targeting moiety disclosed herein comprises at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, or 60% of full length ML-LBP21 without significantly affecting the binding affinity to laminin-2.
  • ML-LBP21 also known histone-like protein/Hlp
  • the targeting moiety disclosed herein comprises a laminin-2-binding domain of ML-LBP21. In some instances, the targeting moiety disclosed herein comprises at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, or 60% of full length ML-LBP21 without significantly affecting the binding affinity to laminin-2.
  • the targeting moiety disclosed herein comprises no greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of full length ML-LBP21 without significantly affecting the binding affinity to laminin-2. In some instances, the targeting moiety disclosed herein comprises a portion of ML-LBP21 with one or more conservative amino acid substitutions without significantly affecting the binding affinity to laminin-2. In some instances, the targeting moiety disclosed herein comprises a portion of ML- LBP21 with one or more non-conservative amino acid substitutions without significantly affecting the binding affinity to laminin-2.
  • the targeting moiety disclosed herein comprises a portion of ML-LBP21 with one or more substitution with a basic, an acidic, an aromatic, an aliphatic, an acid amide, a cyclic, or a sulfur-containing amino acid without significantly affecting the binding affinity to laminin-2. In some instances, the targeting moiety disclosed herein comprises a portion of ML-LBP21 with one or more substitution with a standard amino acid or a non-standard amino acid without significantly affecting the binding affinity to laminin-2.
  • the targeting moiety disclosed herein comprises a portion of ML-LBP21 with one or more substitution with an alpha, a beta, a gamma, or a delta amino acid without significantly affecting the binding affinity to laminin-2. In some instances, the targeting moiety disclosed herein comprises a portion of ML-LBP21 with one or more substitutions with a positively-charged, negatively-charged, or neutral amino acid without significantly affecting the binding affinity to laminin-2. In some instances, the targeting moiety disclosed herein comprises a portion of ML-LBP21 with one or more substitution with a polar or non-polar amino acid without significantly affecting the binding affinity to laminin-2.
  • the targeting moiety disclosed herein comprises a portion of ML-LBP21 with one or more amino acid deletions without significantly affecting the binding affinity to laminin- 2. In some instances, the targeting moiety disclosed herein comprises a portion of ML-LBP21 with one or more amino acid insertions without significantly affecting the binding affinity to laminin-2. In some instances, the targeting moiety disclosed herein comprises a portion of ML- LBP21 and an amino acid sequence which is at least partly the reverse of the amino acid sequence of the corresponding portion of ML-LBP21, without significantly affecting the binding affinity to laminin-2.
  • the targeting moiety disclosed herein comprises a portion of ML-LBP21, and wherein at least one of the amino acids from the original portion of ML-LBP21 is replaced by a stereoisomer (e.g., D-stereoisomer) of that amino acid, without significantly affecting the binding affinity to laminin-2.
  • the targeting moiety disclosed herein comprises a portion of ML-LBP21, and is extended at one or both ends thereof with one or more groups, such as D-amino acids, without significantly affecting the binding affinity to laminin-2.
  • the targeting moiety disclosed herein comprises ML-LBP21 with a combination of one or more of the above-mentioned modifications without significantly affecting the binding affinity to laminin-2.
  • the targeting moiety disclosed herein comprises a recombinant ML- LBP21 peptide or a derivative thereof. In some instances, the targeting moiety disclosed herein comprises a laminin-2-binding domain of recombinant ML-LBP21. In some instances, 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.
  • the targeting moiety disclosed herein comprises no greater than 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% of full length recombinant ML-LBP21 without significantly affecting the binding affinity to laminin-2. In some instances, the targeting moiety disclosed herein comprises a portion of recombinant ML- LBP21 with one or more conservative amino acid substitutions without significantly affecting the binding affinity to laminin-2. In some instances, the targeting moiety disclosed herein comprises a portion of recombinant ML-LBP21 with one or more non-conservative amino acid substitutions without significantly affecting the binding affinity to laminin-2.
  • the targeting moiety disclosed herein comprises a portion of recombinant ML-LBP21 with one or more substitution with a basic, an acidic, an aromatic, an aliphatic, an acid amide, a cyclic, or a sulfur-containing amino acid without significantly affecting the binding affinity to laminin-2. In some instances, the targeting moiety disclosed herein comprises a portion of recombinant ML-LBP21 with one or more substitution with a standard amino acid or a nonstandard amino acid without significantly affecting the binding affinity to laminin-2.
  • the targeting moiety disclosed herein comprises a portion of recombinant ML-LBP21 with one or more substitution with an alpha, a beta, a gamma, or a delta amino acid without significantly affecting the binding affinity to laminin-2. In some instances, the targeting moiety disclosed herein comprises a portion of recombinant ML-LBP21 with one or more substitution with a positively-charged, negatively-charged, or neutral amino acid without significantly affecting the binding affinity to laminin-2. In some instances, the targeting moiety disclosed herein comprises a portion of recombinant ML-LBP21 with one or more substitution with a polar or non-polar amino acid without significantly affecting the binding affinity to laminin-2.
  • the targeting moiety disclosed herein comprises a portion of recombinant ML-LBP21 with one or more amino acid deletions without significantly affecting the binding affinity to laminin-2. In some instances, the targeting moiety disclosed herein comprises a portion of recombinant ML-LBP21 with one or more amino acid insertions without significantly affecting the binding affinity to laminin-2. In some instances, the targeting moiety disclosed herein comprises a portion of recombinant ML-LBP21 and an amino acid sequence which is at least partly the reverse of the amino acid sequence of the corresponding portion of recombinant ML-LBP21 without significantly affecting the binding affinity to laminin-2.
  • the drug molecules disclosed herein modify the expression of a disease- associated molecule, such as modifying expression level of a gene.
  • the overexpression of the disease-associated molecule leads to the disease.
  • the drug molecules disclosed herein reduce the transcription/RNA level of the disease- associated molecule by at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. Accordingly, in some cases, the drug molecules disclosed herein reduce the transcription/RNA level of the disease-associated molecule to a level of those of healthy individuals.
  • the drug molecules disclosed herein reduce the translation/protein level of the disease-associated molecule by at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. Accordingly, in some cases, the drug molecules disclosed herein reduce the translation/protein level of the disease-associated molecule to a level of those of healthy individuals.
  • the lack of the disease-associated molecule leads to the disease.
  • the drug molecules disclosed herein increase the expression level of the disease-associated molecule, such as increase expression level of a gene, for example increase the transcription/RNA level of the disease-associated molecule by at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.
  • the drug molecules disclosed herein increase the transcription/RNA level of the disease-associated molecule to a level of those of healthy individuals.
  • the drug molecules disclosed herein increase the translation/protein level of the disease-associated molecule by at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. Accordingly, in some cases, the drug molecules disclosed herein increase the translation/protein level of the disease-associated molecule to a level of those of healthy individuals.
  • RNA molecules used herein as a drug molecule within the disclosed antibody-drug conjugate are stabilized or modified to improve stabilization and preserve the integrity of RNA.
  • Stabilization and /or modifications of RNA molecules (e.g. siRNA) within the scope of the present invention include, but are not limited to:
  • siRNA small interfering RNA
  • Stabilization of small interfering RNA (siRNA)- Stabilization of siRNA is crucial to improve its stability, bioavailability, and efficacy in therapeutic applications.
  • siRNA molecules are prone to degradation by nucleases, rapid renal clearance, and immune system activation.
  • Several chemical and structural modifications can be employed to stabilize siRNA (see jnanobiotechnology.biomedcentral.com ), including
  • 2'-O-Methyl (2'-0Me) A common modification that improves resistance to nucleases and reduces immunogenicity.
  • LNAs Locked Nucleic Acids
  • Phosphorothioate Linkages Substitute one of the non-bridging oxygen atoms in the phosphate backbone with sulfur, increasing nuclease resistance and improving serum stability.
  • Boranophosphate Replace one of the phosphate oxygens with borane, enhancing stability and activity.
  • Modified bases can enhance the affinity of siRNA for the target mRNA and reduce off- target effects. Examples include methylated bases or incorporation of non-natural bases.
  • Addition of Conjugates Adding lipid moieties (e.g., cholesterol), peptides, or polymers (e.g., polyethylene glycol) to improve cellular uptake and reduce degradation.
  • lipid moieties e.g., cholesterol
  • peptides e.g., peptides
  • polymers e.g., polyethylene glycol
  • PEGylation Attaching polyethylene glycol to siRNA enhances circulation time and reduces renal clearance.
  • Lipid or Polymer Conjugates Facilitate entry into cells and provide steric hindrance against enzymatic degradation.
  • the drug molecules disclosed herein modify the activity of a disease- associated molecule.
  • the hyperactivity of the disease-associated molecule leads to the disease.
  • the drug molecules disclosed herein reduce the activity of the disease-associated molecule by at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.
  • the drug molecules disclosed herein reduce the activity of the disease-associated molecule to a level of those of healthy individuals.
  • the hypoactivity of the disease-associated molecule leads to the disease.
  • the drug molecules disclosed herein increase the activity of the disease-associated molecule by at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. Accordingly, in some cases, the drug molecules disclosed herein increase the activity of the disease-associated molecule to a level of those of healthy individuals.
  • the drug molecule comprises a drug or a precursor form of the drug (a drug precursor or a prodrug).
  • the drug molecule comprises a peptide.
  • the drug molecule comprises a protein. Any suitable peptide or protein may be used as a drug molecule to modify the expression or activity of the disease-associated molecule, as described herein.
  • the drug molecule comprises an enzyme.
  • peptides or proteins may be produced, synthesized, and/or derivatized using several methodologies, e.g. phage displayed peptide libraries, one-bead one-compound peptide libraries, directed evolution, or positional scanning synthetic peptide combinatorial libraries.
  • the drug molecule comprises peptide or protein of about 2-25 amino acids, about 2-20 amino acids, about 2-15 amino acids, about 2-10 amino acids, or about 2-5 amino acids.
  • the peptide or protein may comprise naturally-occurring amino acids, e.g. cysteine, alanine, or non-naturally-occurring or modified amino acids.
  • Non-naturally occurring amino acids include 3 -amino acids, homo-amino acids, proline derivatives, 3 -substituted alanine derivatives, linear core amino acids, N-methyl amino acids, and others known in the art.
  • the peptide may be linear; in other instances, the peptide may be cyclic, e.g. bicyclic.
  • the conjugated drug molecules disclosed herein are small molecular weight (SMW), also referred to as small molecule (SM), compounds that inhibit or modulate the activity of specific enzymes, proteins, kinases and/or phosphatases or any cell activity-related moiety or any protein-specific modulating agent.
  • SMW small molecular weight
  • such enzymes, proteins, kinases and/or phosphatases are implicated in many cellular processes, such as proliferation, differentiation and oncogenic transformation. These proteins are crucial regulators of intracellular signaling pathways mediating multiple cellular activities. Therefore, alterations in kinases and phosphatases functionality may be involved in potency of cancer cells (e.g. metastasis or metastatic disease).
  • SMW inhibitors of kinases include, in a non limiting manner, calcium/calmodulin, phospholipids, cAMP and cGMP, growth factors, cytokines, and toxins.
  • SMW inhibitors of phosphatases include, in a non limiting manner, okadaic acid, calyculin, tautomycin, certain vanadium compounds, and other phosphatase inhibitors.
  • a non limiting list of kinases and phosphatases SMW inhibitors may include, AKT (Protein Kinase B) Inhibitors, MAP Kinase Inhibitors, Protein Kinase C Inhibitors, Protein Phosphatase Inhibitors, Protein Tyrosine Kinase Inhibitors, Rho Kinase (ROCK) Inhibitors and/or any other Kinase/Phosphatase Inhibitors known in the art.
  • AKT Protein Kinase B
  • MAP Kinase Inhibitors Protein Kinase C Inhibitors
  • Protein Phosphatase Inhibitors Protein Tyrosine Kinase Inhibitors
  • Rho Kinase (ROCK) Inhibitors and/or any other Kinase
  • the drug molecule comprises a small molecule.
  • the small molecule is a modulator of the disease-associated molecule.
  • the small molecule is an agonist of the disease-associated molecule.
  • the small molecule is an antagonist of the disease-associated molecule.
  • the small molecule is an inhibitor of the disease-associated molecule.
  • the small molecule is a protein degrader molecule, for targeted protein degradation of a Protein of Interest (e.g. a disease associated molecule), for example, Mancarella, C., et al., Int J Mol Sci., 2023, 24, 16346, is incorporated herein by reference in its entirety.
  • cytotoxic agents include, but are not limited to auristatins (e.g., auristatin e, auristatin f, MMAE and MMAF), auromycins, maytansinoids, calicheamicins, pyrrolobenzodiazepines, topoisomerase inhibitors, a-Amanitin, epothilones, ricin, ricin A- chain, combretastatin, duocarmycins, dolastatins, doxorubicin, daunorubicin, taxols, cisplatin, ccl065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, dihydroxy anthracin dione, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE) A, PE40, abrin, abrin A chain, mode
  • the inventors of the present invention developed a conjugate of a cell death inducing protein (or a cytotoxic agent) with a targeting moiety specific to a cell adhesion moiety or receptor expressed or overexpressed by rapidly dividing cancer cells (e.g. a specific type of cancer cells).
  • the cell death inducing proteins are toxins that are cytopathic to the target cells, e.g. through lytic or non- lytic mechanisms by inducing necrosis or apoptosis.
  • cell death types include apoptosis, autophagy, necrosis, and entosis.
  • toxins that can induce cell death include, but are not limited to, autophagic cell death toxins, including cytostatics, ionophores, and oxidants inducing autophagic cell death; ER stress is known to be able to cause apoptosis as well as autophagic cell death; TCDD and cadmium can induce cell death by either apoptosis or autophagy.
  • cell death is caused by extrinsic pathways, generally initiated by the activation of death receptors through the interaction between their natural ligands or by inducing death receptor clusterization.
  • the intracellular death domain (DD) of death receptors associates with an adaptor protein called Fas-associated death domain (FADD) directly or indirectly via the TNF receptor-associated death domain.
  • the death receptor associated intracellular FADD interacts with pro-caspase-8, a typical initial caspase, to form a death-inducing signaling complex required for caspase- 8 activation.
  • pro-caspase-8 a typical initial caspase
  • cytochrome c which binds to Apaf-1 and promotes caspase-9 and caspase-3 activation.
  • anti-apoptotic Bcl-2 and Bcl-xL protect these effects by maintaining the MTP through the inhibition of Bax or other pro-apoptotic factors.
  • the loss of balance of Bcl-2/Bax is believed to contribute to the progression of apoptosis.
  • the drug molecule comprises an oligonucleotide.
  • oligonucleotide comprises a double-stranded RNAi molecule or a single-stranded antisense oligonucleotide.
  • the drug molecule comprises a small interfering RNA (siRNA), a microRNA (miRNA), an inhibitory double stranded RNA (dsRNA), a small or short hairpin RNA (shRNA), a piwi-interacting RNA (piRNA), a heterogeneous nuclear RNA (hnRNA), a small nuclear RNA (snRNA), or an enzymatically-prepared siRNA (esiRNA) or the precursors thereof.
  • siRNA small interfering RNA
  • miRNA microRNA
  • dsRNA inhibitory double stranded RNA
  • shRNA small or short hairpin RNA
  • piRNA piwi-interacting RNA
  • hnRNA heterogeneous nuclear RNA
  • snRNA small nuclear
  • the disease-associated molecule is antiganglioside antibodies.
  • the drug molecule disclosed herein modifies antiganglioside antibodies.
  • antiganglioside antibodies are associated with the Guillain-Barre syndrome.
  • the Guillain-Barre syndrome is a post-infectious immune-mediated neuropathy, which results from the autoimmune destruction of nerves in the peripheral nervous system.
  • the syndrome may include muscle movement as well as those that transmit pain, temperature and touch sensations. This can result in muscle weakness and loss of sensation in the legs and/or arms.
  • Antiganglioside antibodies may be involved in the pathogenesis of the Guillain-Barre syndrome.
  • the disease-associated molecule is a subunit of SWI/SNF protein complexes, which is encoded by gene SMARCB1.
  • the disease-associated molecule is LZTR1 protein, which is encoded by LZTR1 gene.
  • the drug molecule disclosed herein modifies the subunit of SWI/SNF protein complexes.
  • the drug molecule disclosed herein modifies LZTR1 protein.
  • the subunit of SWI/SNF protein complexes is associated with schwannomatosis.
  • LZTR1 protein is associated with schwannomatosis.
  • Schwannomatosis is a type of neurofibromatosis that causes multiple nerve sheath tumors. It is within the scope of the present invention that usage of a small molecule as a drug molecule within the drug conjugate as herein disclosed is provided to target disease associated molecules of Schwannomatosis.
  • the drug molecule is immunoglobulin
  • the associated disease is chronic inflammatory demyelinating polyneuropathy (CIDP).
  • CIDP is a rare disorder of the peripheral nerves characterized by gradually increasing symmetrical motor and sensory loss and weakness associated with loss of deep tendon reflexes. It is caused by damage to Schwann cells. The gradual onset of CIDP can delay diagnosis by several months or even years, resulting in significant nerve damage that may limit and delay the response to therapy. Most individuals may require long term treatment; nearly a third of CIDP patients may progress to wheelchair dependence if left untreated. Early recognition and proper treatment are critical in helping patients avoid a significant amount of disability.
  • the drug molecules of the present invention are small molecules that can modulate the course of the disease of Neurofibromas.
  • Neurofibromas which are the most common peripheral nerve sheath tumor and are often found either by patients or during routine skin exams. These lesions appear as soft, skin-colored papules or small subcutaneous nodules.
  • neurofibromas There are three main types of neurofibromas: localized, diffuse, and plexiform. The majority of neurofibromas are sporadically-occurring and localized and have an extremely low risk of malignant transformation. However, the plexiform type is pathognomonic for neurofibromatosis type 1 and carries an increased risk of malignant transformation. Literature suggests the contribution of ErbB family receptors in neurofibroama progression.
  • RTKs transmembrane receptor tyrosine kinases
  • ErbB Epidermal growth factor receptor family B
  • EGFR epidermal growth factor receptor family B
  • ErbB2 ErbB2
  • ErbB3 ErbB3
  • Uncontrolled RTK signals lead to cell growth disorders and cancer, which provided the rationale for the development of strategies for the prevention and interception of RTK signaling as a way to treat Neurofibromas.
  • An additional number of RTKs have been shown to be associated with neurofibromas, such platelet-derived growth factor (PDGF), fibroblasts growth factor (FGF), insulin-like growth factor- 1, and vascular endothelial growth factor (VEGF).
  • PDGF platelet-derived growth factor
  • FGF fibroblasts growth factor
  • VEGF vascular endothelial growth factor
  • nrPTKs including Src, BCR-ABL, and the JAK/STAT pathway
  • Src Src
  • BCR-ABL BCR-ABL
  • JAK/STAT pathway JAK/STAT pathway
  • nrPTKs are not receptors per se. They generally do not have an extracellular domain, but are coupled to the cell membrane or exist in the cytoplasm, and their tyrosine kinase activity can be activated after binding to the activated receptor.
  • the activation of nrPTKs and downstream signal transduction pathways can promote cell proliferation, cell apoptosis resistance, and oncogenesis. Further, over-activation of the Src pathway has been found in NF2. Therefore, direct and specific SMW compound inhibition of these kinase domain can serve as potential therapeutic pathway to neurofibromas.
  • the targeting moieties disclosed herein can be coupled or conjugated to the drug molecules disclosed herein directly (e.g., through a covalent or non- covalent linkage), or through a linker (e.g., a cleavable linker versus a non-cleavable linker, or a peptide linker versus a non-peptide linker), or through an amino acid or other functional group.
  • a linker e.g., a cleavable linker versus a non-cleavable linker, or a peptide linker versus a non-peptide linker
  • linker may generally refers to a compound, or pharmaceutically acceptable solvate, stereoisomer, or derivative thereof, comprising a binding agent linked to at least one pay load moiety (or drug molecule) via e.g. a covalent linker, wherein the said covalent linker is bonded directly or indirectly to each of the binding agent and the payload moiety.
  • binding agent refers to a bioconjugation moiety or area.
  • Bioconjugation generally means a specialized area of chemistry that focuses on linking biomolecules to one another or to other types of molecules, such as drugs, fluorescent probes, or nanoparticles. It is herein acknowledged that this process is integral to various fields, including diagnostics, therapeutics, and biomaterials development. Effective bioconjugation relies on reactions that are highly specific, operate under mild conditions to preserve the biomolecule's function, and result in stable products suitable for biological environments. Common functional groups targeted in bioconjugation include primary amines in lysine residues, thiols in cysteine residues, carboxyl groups, and hydroxyl groups.
  • maleimides are widely used to selectively react with thiols, forming stable thioether bonds.
  • Other strategies include click chemistry, which involves azide-alkyne cycloaddition for bio- orthogonal labeling, and PEGylation, where polyethylene glycol is attached to proteins or drugs to enhance their solubility and stability (see for example Kalia., J et.al. Curr. Org. Chem, 2010 Jan; 14(2): 138-147 doi: 10.2174/138527210790069839, incorporated by its entirety).
  • the linker is a cleavable linker, a non-cleavable linker, covalently bound linker such as by chemical conjugation, linkers based on non-covalent interactions, a carbohydrate linker, a peptide linker, a glycan linker, or a nonpeptide linker.
  • linkers examples include, but not limited to, Hydrazone Linkers, Disulfide Linkers, Peptide Linkers (Valine-citrulline (Val-Cit) and Alanine-alanine (Ala- Ala)), Beta-Glucuronide Linkers, Thioether Linkers (such as SMCC (succinimidyl-4-(N- maleimidomethyl)cyclohexane-l -carboxylate)), PEG Linkers, Conditional Linkers (such as Azobenzene Linkers and Diselenide Linkers).
  • the targeting moieties disclosed herein and the drug molecules disclosed herein are connected via a non-covalent interaction.
  • the non- covalent interaction comprises a biotin/avidin interaction.
  • Examples of connecting connect the targeting moiety and the drug molecule include, but are not limited to, the use of biotin and avidin or streptavidin (see, e.g., U.S. Pat. No. 4,885,172 A), typical biotin/avidin alternatives (e.g., FITC/anti-FITC (see, e.g., Harmer and Samuel (1989) J. Immunol. Meth. 122(1): 115- 221), di oxigenin/anti-di oxigenin, and the like.
  • the targeting moieties disclosed herein and the drug molecules disclosed herein are connected via a covalent interaction.
  • the targeting moieties disclosed herein and the drug molecules disclosed herein are connected via chemical conjugation.
  • Traditional chemical conjugation using, for example, bifunctional coupling agents such as glutaraldehyde, diimide esters, aromatic and aliphatic diisocyanates, bis-p-nitrophenyl esters of dicarboxylic acids, aromatic disulfonyl chlorides and bifunctional arylhalides such as l,5-difluoro-2,4-dinitrobenzene; p,p’-difluoro m,m’ -dinitrodiphenyl sulfone, sulfhydrylreactive maleimides, and the like.
  • the chemical conjugation of the targeting moieties disclosed herein and the drug molecules disclosed herein is via a non-cleavable linker or via a cleavable linker.
  • the cleavable linkers are generally cleavable only intracellularly and are preferably stable in extracellular environments, e.g. extracellular to the Schwann cells.
  • the cleavable linker disclosed herein comprises a chemically cleavable linkers.
  • the cleavable linker disclosed herein comprises an enzymatically cleavable linker.
  • the chemically cleavable linker is pH-sensitive linker.
  • the pH-sensitive linker disclosed herein is readily degrades in high pH environments.
  • the pH-sensitive linker disclosed herein is readily degrades in low pH environments.
  • the pH-sensitive linker may be cleaved at a pH in a range of 4 to 6.
  • the pH-sensitive linker comprises a hydrazone or cyclic acetal.
  • the pH-sensitive linker is cleaved within an endosome or a lysosome.
  • the chemically cleavable linker is glutathione-sensitive linker.
  • the glutathione-sensitive linker is cleaved by an disulfide exchange reaction with a glutathione species inside a cell.
  • the disulfide moiety further comprises at least one amino acid, e.g. a cysteine residue.
  • Other illustrative chemically cleavable linkers include, but are not limited to, acid-labile linkers, disulfide linkers, and the like.
  • Acid-labile linkers are designed to be stable at pH levels encountered in the blood, but become unstable and degrade when the low pH environment in lysosomes is encountered, an acid-labile linker comprising a moiety selected from the group consisting of a hydrazone, an acetal, a cis-aconitate amide, and a silyl ether.
  • Acid-sensitive linkers include, but are not limited to hydrazones, acetals, cis-aconitate-like amides, and silyl ethers (see, e.g., Perez et al. (2013) Drug Discov. Today, 1-13).
  • Hydrazones are easily synthesized and have a plasma half-life of 183 hours at pH 7 and 4.4 hours at pH 5, indicating that they are selectively cleavable under acidic conditions such as those found in the lysosome (see, e.g., Doronina et al. 92013) Nat. Biotechnol. 21(7): 778-784).
  • Disulfide bridges are cleavable linkers that take advantage of the cellular reducing environment (see, e.g., Saito et al. (2013) Adv. Drug Deliv. Rev. 55(2): 199-215). After internalization and degradation, disulfide bridges can release drugs in the lysosome.
  • Enzymatically cleavable linkers are selected to be cleaved by an enzyme (e.g., a protease).
  • an enzyme e.g., a protease
  • protease-cleavable linkers are typically designed to be stable in blood/plasma, but are rapidly cleaved in lysosomes by lysosomal enzymes.
  • the most popular enzymatic cleavage sequence is the dipeptide valine-citrulline, combined with a self- immolative linker p-aminobenzyl alcohol (PAB).
  • the cleavable linker is a protease-sensitive linker.
  • the protease-sensitive linker described herein may comprise a sequence cleavable by a lysosomal protease and/or an endosomal protease.
  • the protease-sensitive linker typically comprises peptide sequences and may be 2-10 amino acids, about 2-5 amino acids, about 5-10 amino acids, about 10 amino acids, about 5 amino acids, about 3 amino acids, or about 2 amino acids in length.
  • the protease-sensitive linker comprises naturally-occurring amino acids, e.g. cysteine, alanine, or non-naturally-occurring or modified amino acids.
  • Non-naturally occurring amino acids include 3-amino acids, homo-amino acids, proline derivatives, 3-substituted alanine derivatives, linear core amino acids, N-methyl amino acids.
  • the proteasesensitive linker comprises a valine-citrulline or alanine-citrulline dipeptide sequence.
  • the protease-sensitive linker can be cleaved by a lysosomal protease, e.g. cathepsin B, and/or an endosomal protease.
  • the targeting moiety and the drug molecule are covalently linked to each other via a non-cleavable linker.
  • the non-cleavable linker cannot be readily degraded in a cellular or physiological environment.
  • the non-cleavable linker described herein comprises an optionally substituted alkyl group, wherein the substitutions may include halogens, hydroxyl groups, oxygen species, and other common substitutions.
  • the non-cleavable linker described herein comprises an optionally substituted alkyl, an optionally substituted alkylene, an optionally substituted arylene, a heteroarylene, a peptide sequence comprising at least one non-natural amino acid, a truncated glycan, a sugar or sugars that cannot be enzymatically degraded, an azide, an alkyne-azide, a peptide sequence comprising a LPXT sequence, a thioether, a biotin, a biphenyl, repeating units of polyethylene glycol or equivalent compounds, acid esters, acid amides, sulfamides, and/or an alkoxy-amine linker.
  • sortase-mediated ligation is utilized to covalently link the drug molecule comprising a LPXT sequence to the targeting moiety comprising a (G).
  • sortase-mediated ligation is utilized to covalently link the targeting moiety comprising a LPXT sequence to the drug molecule comprising a (G)sequence (see, e.g. Proft T. Sortase-mediated protein ligation: an emerging biotechnology tool for protein modification and immobilization. Biotechnol Lett. 2010, 32(1): 1-10, which is incorporated by reference herein in its entirety).
  • the non-cleavable linkers described herein comprises a substituted alkylene, an optionally substituted alkenylene, an optionally substituted alkynylene, an optionally substituted cycloalkylene, an optionally substituted cycloalkenylene, an optionally substituted arylene, an optionally substituted heteroarylene further comprising at least one heteroatom selected from N, O, and S; an optionally substituted heterocyclylene further comprising at least one heteroatom selected from N, O, and S; an imino, an optionally substituted nitrogen species, an optionally substituted oxygen species O, an optionally substituted sulfur species, or a poly(alkylene oxide), e.g. polyethylene oxide or polypropylene oxide.
  • a poly(alkylene oxide) e.g. polyethylene oxide or polypropylene oxide.
  • the linkers described herein are peptide linkers.
  • the peptide linker is relatively short, typically about 20 amino acids or less, about 15 amino acids or less, about 10 amino acids or less, about 8 amino acids or less, about 5 amino acids or less, about 3 amino acids or less, or is a single amino acid.
  • the peptide linker disclosed herein comprises an amino acid sequence cleavable by a protease.
  • the peptide linker disclosed herein comprises an amino acid sequence cleavable by a cathepsin.
  • the peptide linker disclosed herein comprises a dipeptide valine-citrulline (Val-Cit) (see U.S. Pat. No.
  • Val-Cit showed a half-life of 240 min. They also found that removal of the PAB group reduced the cleavage rate, presumably through steric interference with enzyme binding.
  • Another study compared the potency of auristatin derivative MMAE linked by dipeptide linkers Phe-Lys and Val-Cit and an analogous hydrazone linker.
  • the Val-Cit linker proved to be over 100 times as stable as the hydrazone linker in human plasma. Most significantly, the Phe-Lys linker was substantially less stable than Val-Cit in human plasma, which accounts for its current popularity (see, e.g., Doronina et al. (2003) Nat. Biotechnol. 21(7): 778-784).
  • peptide linkers include, but not limited to, alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, GGGG, PSGSP, PSPSP, KKKK, RRRR, ASASA, GGSGGS, GGGGS, GGGGS GGGGS, GGGGS GGGGS GGGGS GGGGS, GGGGS GGGGS GGGGS GGGGS GGGGS, GGGGS GGGGS GGGGS GGGGS GGGGS, GGGGS GGGGS GGGGS GGGGS FK GGGGS GGGGS GGGGS, and GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS GGGGS FK GGGGS GGGGS GGGGS, and GGGGS GGG
  • the linkers described herein utilize PASYLATION®-technology, which is a genetic fusion with conformationally disordered polypeptide sequences composed of the amino acids Pro, Ala, and/or Ser.
  • the linkers described herein are selected from the ones disclosed in patents such as U.S. Patent 10,961,287, which is incorporated by reference herein in its entirety.
  • the linkers described herein are non-peptide linkers.
  • a glucuronide linker incorporates a hydrophilic sugar group that is cleaved by the lysosomal enzyme beta glucuronidase. Once the sugar is cleaved from the phenolic backbone, self-immolation of the PAB group releases the conjugated moiety (see, e.g., Jeffrey et al. (2006) Bioconjug. Chem. 17(3): 831-840).
  • the linkers described herein are derived from natural multidomain proteins (see, e.g., Chen at al., (2013) Adv Drug Deliv Rev. 65(10): 1357-1369).
  • the targeting moieties disclosed herein may be attached to the drug molecules disclosed herein via certain functional groups or via certain reactions.
  • the linker disclosed herein comprises a functional group that is reactive with a corresponding functional group on the targeting moieties disclosed herein and/or the drug molecules disclosed herein.
  • a bifunctional linker has one functional group reactive with a group on the targeting moieties disclosed herein and another functional group reactive on the drug molecules disclosed herein and can be used to form the desired conjugate.
  • a heterobifunctional linker comprises two or more different reactive groups that react with sites on the targeting moieties disclosed herein and the drug molecules disclosed herein, respectively.
  • a heterobifunctional crosslinker such as cysteine may comprise an amine reactive group and a thiol-reactive group can interact with an aldehyde on a derivatized peptide.
  • Additional combinations of reactive groups suitable for heterobifunctional crosslinkers include, for example, amine- and sulfhydryl reactive groups; carbonyl and sulfhydryl reactive groups; amine and photoreactive groups; sulfhydryl and photoreactive groups; carbonyl and photoreactive groups; carboxylate and photoreactive groups; and arginine and photoreactive groups.
  • Such reactions and functional groups are illustrative and non-limiting.
  • Suitable reactive groups include, but are not limited to thiol ( — SH), carboxylate (COOH), carboxyl ( — COOH), carbonyl, amine (NH2), hydroxyl ( — OH), aldehyde ( — CHO), alcohol (ROH), ketone (R2CO), active hydrogen, ester, sulfhydryl (SH), phosphate ( — PO3), or photoreactive moieties.
  • Amine reactive groups include, but are not limited to e.g., isothiocyanates, isocyanates, acyl azides, NHS esters, sulfonyl chlorides, aldehydes and glyoxals, epoxides and oxiranes, carbonates, arylating agents, imidoesters, carbodiimides, and anhydrides.
  • Thiol-reactive groups include, but are not limited to e.g., haloacetyl and alkyl halide derivates, maleimides, aziridines, acryloyl derivatives, arylating agents, and thiol-disulfides exchange reagents.
  • Carboxylate reactive groups include, but are not limited to e.g., diazoalkanes and diazoacetyl compounds, such as carbonyldiimidazoles and carbodiimides.
  • Hydroxyl reactive groups include, but are not limited to e.g., epoxides and oxiranes, carbonyldiimidazole, oxidation with periodate, N,N'-disuccinimidyl carbonate or N- hydroxylsuccimidyl chloroformate, enzymatic oxidation, alkyl halogens, and isocyanates.
  • the linkers described herein connect the targeting moiety to the drug molecule by a cycloaddition reaction between an azide and an alkyne to form a triazole, wherein the azide and the alkyne may be located on the targeting moiety, the drug molecule, or the linker.
  • an alkyne may be a cyclic alkyne, e.g., a cyclooctyne.
  • an alkyne may be bicyclononyne (also known as bicyclononyne or BCN) or substituted bicyclononyne.
  • an electrophile may exist on a linker and a nucleophile may exist on the targeting moiety or the drug molecule prior to a reaction between a linker and the targeting moiety or the drug molecule.
  • an electrophile may be an azide, a silicon centers, a carbonyl, a carboxylic acid, an anhydride, an isocyanate, a thioisocyanate, a succinimidyl ester, a sulfosuccinimidyl ester, a maleimide, an alkyl halide, an alkyl pseudohalide, an epoxide, an episulfide, an aziridine, an aryl, an activated phosphorus center, and/or an activated sulfur center.
  • the linkers described herein further comprises a spacer, e.g., a polyethylene glycol spacer or an acyl/carbomoyl sulfamide spacer, e.g., a HydraSpaceTM spacer.
  • a spacer is as described in Verkade, J. M. M. et al., “A Polar Sulfamide Spacer Significantly Enhances the Manufacturability, Stability, and Therapeutic Index of Antibody-Drug Conjugates”, Antibodies, 2018, 7, 12, which is incorporated by reference herein in its entirety.
  • the linkers disclosed herein have no specific biological activity other than to join the proteins or to preserve some minimum distance or other spatial relationship between them.
  • the constituent amino acids of the spacer may be selected to influence some property of the molecule such as the folding, net charge, or hydrophobicity.
  • the linker may comprise an enzymatic cleavage site.
  • one of the targeting moiety and drug molecule is a peptide/protein or an antibody or an antigen binding fragment thereof and the other is a nucleotide.
  • the peptide/protein or an antibody or an antigen binding fragment thereof comprises a nonnatural amino acid to which the nucleotides can be covalently linked.
  • the peptide/protein or an antibody or an antigen binding fragment thereof is covalently linked to the nucleotide via conjugation to a lysine residue or a cysteine residue of the peptide/protein or an antibody or an antigen binding fragment thereof.
  • the nucleotide is conjugated to a cysteine residue of the peptide/protein or an antibody or an antigen binding fragment thereof via a maleimide-containing linker, optionally wherein the maleimide-containing linker comprises a maleimidocaproyl or maleimidomethyl cyclohexane- 1 -carboxylate group.
  • peptide/protein or an antibody or an antigen binding fragment thereof is glycosylated that comprises at least one sugar moiety to which the nucleotide is covalently linked.
  • the at least one sugar moiety comprises at least one sugar moiety that is a branched mannose.
  • the peptide/protein or an antibody or an antigen binding fragment thereof is glycosylated that comprises more than one sugar moieties, and each of all or a portion of the more than one sugar moieties is covalently linked to a separate nucleotide.
  • the peptide/protein or an antibody or an antigen binding fragment thereof is a fully-glycosylated or a partially-glycosylated.
  • a partially-glycosylation may be produced via chemical or enzymatic means.
  • a partially-glycosylation is produced in a cell that is deficient for an enzyme in the N- or O-glycosylation pathway.
  • the linkers described herein are connected to the peptide/protein or an antibody or an antigen binding fragment thereof via a phosphate, thioether, ether, carbon-carbon, or amide bond. In some instances, the linkers described herein are connected to the nucleotide through a phosphate or phosphorothioate group, e.g. a terminal phosphate of an oligonucleotide backbone.
  • both the targeting moiety and drug molecule are peptides/proteins or antibodies or antigen binding fragments thereof. Accordingly, in some cases, the targeting moiety and drug molecule can be expressed as a fusion protein. In some specific cases, the targeting moiety and drug molecule are directly attached to each other. In some specific cases, the targeting moiety and drug molecule are connected via an intervening amino acid. In some specific cases, the targeting moiety and drug molecule are connected through a peptide linker.
  • the linkers can be joined to the constituent amino acids through their side groups (e.g., through a disulfide linkage to cysteine), while in other instances, the linkers are joined to the alpha carbon amino and carboxyl groups of the terminal amino acids when such are present.
  • This involves creating a DNA sequence that encodes the fusion protein, placing the DNA in an expression cassette under the control of a particular promoter, expressing the protein in a host, isolating the expressed protein and, if required, renaturing the protein.
  • DNA encoding the fusion proteins can be prepared by any suitable method, including, for example, cloning and restriction of appropriate sequences or direct chemical synthesis by methods such as the phosphotriester method of Narang et al. (1979) Meth. Enzymol. 68: 90-99; the phosphodiester method of Brown et al. (1979) Meth. Enzymol. 68: 109-151; the diethylphosphoramidite method of Beaucage et al. (1981) Tetra. Lett., 22: 1859-1862; and the solid support method ofU.S. Pat. No. 4,458,066.
  • DNA encoding fusion proteins constructs described herein may be cloned using DNA amplification methods such as polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • the nucleic acid sequence encoding the targeting moieties disclosed herein can be PCR amplified, using primers containing the designed restriction sites. This produces a nucleic acid encoding the targeting moieties disclosed herein and having terminal restriction sites.
  • the nucleic acid encoding the drug molecules disclosed herein can be provided having complementary restriction sites. Ligation of sequences and insertion into a vector produces a vector encoding the fusion protein.
  • the nucleic acid sequences encoding the fusion proteins can be expressed in a variety of host cells, including E.
  • the recombinant protein gene will be operably linked to appropriate expression control sequences for each host.
  • this includes a promoter such as the T7, trp, or lambda promoters, a ribosome binding site and preferably a transcription termination signal.
  • the control sequences will include a promoter and preferably an enhancer derived from immunoglobulin genes, SV40, cytomegalovirus, etc., and a polyadenylation sequence, and may include splice donor and acceptor sequences.
  • the plasmids can be transferred into the chosen host cell by well-known methods such as calcium chloride transformation for E. coli and calcium phosphate treatment or electroporation for mammalian cells.
  • Cells transformed by the plasmids can be selected by resistance to antibiotics conferred by genes contained on the plasmids, such as the amp, gpt, neo and hyg genes.
  • the recombinant fusion proteins can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like (see, generally, R. Scopes (1982) Protein Purification, Springer- Verlag, N.Y.; Deutscher (1990) Methods in Enzymology Vol. 182: Guide to Protein Purification., Academic Press, Inc. N.Y.). Substantially pure compositions of at least about 90 to 95% homogeneity are preferred, and 98 to 99% or more homogeneity are most preferred for pharmaceutical uses. Once purified, partially or to homogeneity as desired, the polypeptides may then be used therapeutically.
  • modifications can be made to the fusion proteins without diminishing their biological activity. Some modifications may be made to facilitate the cloning, expression, or incorporation of the drug molecules or targeting moieties disclosed herein into a fusion protein. Such modifications include, for example, a methionine added at the amino terminus to provide an initiation site, or additional amino acids placed on either terminus to create conveniently located restriction sites or termination codons.
  • one of the targeting moiety and drug molecule is a peptide/protein or an antibody or an antigen binding fragment thereof and the other is a small molecule.
  • the exemplary linkers can be found in U.S. Patents 10864279, 9814784, 11173214, 11104968, 9872924, 10537644, U.S. patent applications US20180169262, US20220072137, and US20180311375, which are incorporated by reference herein in their entireties.
  • the drug conjugate is an AOC (antibody oligonucleotide conjugate) comprising a fatty acid conjugated oligonucleotide.
  • the targeting moiety is a fatty acid (or a fatty acid ligand) and the drug molecule is an oligonucleotide (e.g. siRNA).
  • a drug conjugate is useful for treating neuroscience, neuromuscular and CNS indications e.g., neuromuscular disorder Charcot-Marie-Tooth (CMT) disease, such as CMT type 1A (CMT1A), axonal loss, dysfunction of the neuronal axon, or abnormal axon-Schwann cell interaction.
  • CMT neuromuscular disorder Charcot-Marie-Tooth
  • fatty acid receptors conjugate enable cellular uptake and fatty acid albumin interactions to promote biodistribution of RNA therapeutics beyond the liver (extrahepatic delivery). For example, it decreases the expression of PMP22 protein in Schwann cells, which is the target cell type for the development, maintenance, and function of peripheral nerves.
  • the fatty acid conjugated oligonucleotide facilitates the targeted approach of the present invention by conjugating siRNAs to naturally occurring fatty acids to improve the biodistribution and cellular uptake to tissues and cell types of interest.
  • multiple targeting moieties disclosed herein can be attached to a single drug molecule disclosed herein.
  • multiple the drug molecules disclosed herein can be attached to a single targeting moiety disclosed herein.
  • a single targeting moiety disclosed herein is attached to a single drug molecule disclosed herein.
  • the Drug molecule to the Targeting Moiety Ratio is 1 to 10 or any ratio between 2 to 10.
  • the Drug-Targeting Moiety Ratio as measured according to Matsuda, Y., Mendelsohn, BA., Chem Pharm Bull (Tokyo). 2021 ;69, 976, is between 2 to 8.
  • compositions comprising drug conjugates disclosed herein and pharmaceutically acceptable excipients.
  • the pharmaceutical compositions are formulated for parenteral, intravenous, subcutaneous, intrathecal, or intrasciatic injection.
  • the drug conjugate provided herein preferably internalizes to Schwann cells. In some instances, the drug conjugate provided herein internalizes to Schwann cells at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 fold(s) more than it internalizes to a cell type other than Schwann cells.
  • the drug conjugate provided herein enters blood stream and stays intact. In some instances, the drug conjugate provided herein enters blood stream and effectively crosses the blood nerve barrier. In some instances, the drug conjugate provided herein enters blood stream, effectively crosses the blood nerve barrier, and enriches in sciatic nerves.
  • the pharmaceutical composition described herein can be prepared to include the drug conjugates disclosed herein, in a form suitable for administration to a subject using carriers, excipients, and vehicles.
  • excipients include magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talc, milk protein, gelatin, starch, vitamins, cellulose and its derivatives, animal and vegetable oils, polyethylene glycols and solvents, such as sterile water, alcohols, glycerol, and polyhydric alcohols.
  • Intravenous vehicles include fluid and nutrient replenishers.
  • Preservatives include antimicrobial, anti-oxidants, chelating agents, and inert gases.
  • compositions include aqueous solutions, nontoxic excipients, including salts, preservatives, buffers and the like, as described, for instance, in Remington: The Science and Practice of Pharmacy, 21st Ed., Gennaro, Ed., Lippencott Williams & Wilkins (2005), and The United States Pharmacopeia: The National Formulary (USP 36 NF31), published in 2013. The pH and exact concentration of the various components of the pharmaceutical composition are adjusted according to routine skills in the art. See Goodman and Gilman's, The Pharmacological Basis for Therapeutics.
  • compositions described herein may be administered locally or systemically.
  • the therapeutically effective amounts will vary according to factors, such as the degree of infection in a subject, the age, sex, and weight of the individual. Dosage regimes can be adjusted to provide the optimum therapeutic response. For example, several divided doses can be administered daily or the dose can be proportionally reduced as indicated by the exigencies of the therapeutic situation.
  • the pharmaceutical composition can be administered in a convenient manner, such as by injection (e.g., subcutaneous, intravenous, intraorbital, intrasciatic, intrathecal, and the like), oral administration, ophthalmic application, inhalation, topical application, or rectal administration.
  • the pharmaceutical composition can be coated with a material to protect the pharmaceutical composition from the action of enzymes, acids, and other natural conditions that may inactivate the pharmaceutical composition.
  • the pharmaceutical composition can also be administered parenterally or intraperitoneally.
  • Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof, and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
  • the composition will typically be sterile and fluid to the extent that easy syringability exists.
  • the composition will be stable under the conditions of manufacture and storage and preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • the vehicle can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size, in the case of dispersion, and by the use of surfactants.
  • a coating such as lecithin
  • surfactants Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride are used in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the pharmaceutical composition in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the pharmaceutical composition into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the pharmaceutical composition is formulated in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of pharmaceutical composition is calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle.
  • the specification for the dosage unit forms are related to the characteristics of the pharmaceutical composition and the particular therapeutic effect to be achieve.
  • the principal pharmaceutical composition is compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable vehicle in an acceptable dosage unit. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the ingredients.
  • the pharmaceutical composition can be orally administered, for example, in a carrier, e.g., in an enteric-coated unit dosage form.
  • a carrier e.g., in an enteric-coated unit dosage form.
  • the pharmaceutical composition and other ingredients can also be enclosed in a hard or soft-shell gelatin capsule or compressed into tablets.
  • the pharmaceutical composition can be incorporated with excipients and used in the form of ingestible tablets, troches, capsules, pills, wafers, and the like.
  • the percentage of the compositions and preparations can, of course, be varied and can conveniently be between about 5% to about 80% of the weight of the unit.
  • the tablets, troches, pills, capsules, and the like can also contain the following: a binder, such as gum tragacanth, acacia, corn starch, or gelatin; excipients such as dicalcium phosphate; a disintegrating agent, such as corn starch, potato starch, alginic acid, and the like; a lubricant, such as magnesium stearate; and a sweetening agent, such as sucrose, lactose or saccharin, or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring.
  • a binder such as gum tragacanth, acacia, corn starch, or gelatin
  • excipients such as dicalcium phosphate
  • a disintegrating agent such as corn starch, potato starch, alginic acid, and the like
  • a lubricant such as magnesium stearate
  • a sweetening agent such as sucrose, lactose or saccharin, or a flavoring
  • suitable pharmaceutically acceptable salts include (i) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine, etc.; (ii) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, and the like; and (iii) salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic
  • the drug molecule disclosed herein is a protein degrader molecule.
  • the protein degrader molecule may be conjugated to a targeting moiety for targeted delivery of the protein degrader molecule, achieving targeted and regulated protein degradation of the target molecule (Protein of Interest) such as PROTAC technology (see Mancarella, C., et al., Int J Mol Sci., 2023, 24, 16346, incorporated herein by reference in its entirety) or (ubiquitin-or ubiquitin-like proteasome machinery).
  • kits for targeted delivery of drugs to Schwann cells by contacting the drug conjugates disclosed herein or the pharmaceutical compositions comprising the drug conjugates disclosed herein to the Schwann cells.
  • the drug conjugates are internalized into the Schwann cells upon binding of the targeting moieties to the cell surface molecule expressed on the Schwann cells.
  • the cell surface molecule expressed on the Schwann cells is a cell surface receptor having an extracellular domain.
  • the cell surface molecules are enriched on the Schwann cell surface.
  • the drug conjugates are delivered to the Schwann cells by systemic delivery of the drug conjugates disclosed herein or the pharmaceutical compositions disclosed herein to the body of a subject.
  • the drug conjugates disclosed herein can penetrate the blood nerve barrier. In some instances, the drug conjugates disclosed herein can penetrate through the blood nerve barrier and can be enriched in sciatic nerves. In some aspects, provided herein are methods of preferable delivery of drugs to or into the Schwann cells, by contacting the drug conjugates disclosed herein or the pharmaceutical compositions comprising the drugs disclosed herein to the Schwann cells. In some instances, the drug conjugates are preferably internalized into the Schwann cells upon binding of the targeting moieties to the cell surface molecule expressed on the Schwann cells. In some instances, the cell surface molecule preferably expressed on the Schwann cells is a cell surface receptor having an extracellular domain. In some instances, the cell surface molecules are preferably expressed on the Schwann cell surface.
  • the cell surface molecules are expressed at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 2, 3, 4, 5 folds more in Schwann cells than other types of cells.
  • the methods provided herein ensure that the drugs are preferably internalized into the Schwann cells at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 2, 3, 4, 5, 6, 7, 8, 9, or 10 fold(s) more than being internalized into a cell type other than Schwann cells.
  • the drug conjugates are delivered to the Schwann cells by systemic delivery of the drug conjugates disclosed herein or the pharmaceutical compositions disclosed herein to the body of a subject.
  • the drug conjugates disclosed herein can penetrate the blood nerve barrier.
  • the drug conjugates disclosed herein can penetrate through the blood nerve barrier and can be enriched in sciatic nerves.
  • the methods of targeted delivery disclosed herein possess improved pharmacokinetics.
  • the methods of targeted delivery disclosed herein result in a higher Cmax (i.e., maximum concentration of the drug molecule in Schwann cells).
  • the methods of targeted delivery disclosed herein result in a shorter Tmax when Cmax is reached in Schwann cells.
  • the methods of targeted delivery disclosed herein result in a higher area under the concentration-time curve.
  • the methods of targeted delivery disclosed herein result in a longer ti/2 (i.e., the time taken for the concentration of the drug molecule to fall by one half once distribution equilibrium has been reached in Schwann cells).
  • the methods of targeted delivery disclosed herein result in the circulation and availability of the drug conjugate in the subject for at least 1, 5, 10, 15, 20, 25, 30, 35, or 40 days. In some cases, the methods of targeted delivery disclosed herein result in the circulation and availability of the drug conjugate in the subject for about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120 days. In some cases, the methods of targeted delivery disclosed herein result in the circulation and availability of the drug conjugate in the subject for at most 90, 120, 150, 180, 210, or 240 days.
  • the methods of targeted delivery disclosed herein result in reduced distribution in any other tissues, organs, or cell types than Schwann cells. In some instances, the methods of targeted delivery disclosed herein result in an insignificant amount or a minimum amount in any other tissues, organs, or cell types than Schwann cells. In some instances, the methods of targeted delivery disclosed herein are non-immunogenic. In some instances, the methods of targeted delivery disclosed herein are biodegradable. In some instances, the methods of targeted delivery disclosed herein are applicable for a varieties of different drug molecules.
  • the methods of targeted delivery are achieved by parenteral, intravenous, subcutaneous, intrathecal, or intrasciatic injection of the pharmaceutical compositions disclosed herein. In some instances, the methods of targeted delivery are achieved by intravenous injection of the pharmaceutical compositions disclosed herein. In some instances, the methods of targeted delivery are achieved by topical delivery (see, e.g., Pitiot et al. (2022) Antibodies (Basel) 11(3):56.)
  • kits for treating or preventing a peripheral demyelinating disease or a peripheral neuropathy, or alleviating or reducing symptoms of the peripheral demyelinating disease or the peripheral neuropathy in a subject in need thereof comprising: providing the drug conjugates disclosed herein or the pharmaceutical compositions disclosed herein; and administering the subject the drug conjugates or the pharmaceutical compositions to treat the peripheral demyelinating disease or the peripheral neuropathy, to alleviate or reduce symptoms of the peripheral demyelinating disease or the peripheral neuropathy.
  • the peripheral demyelinating disease or the peripheral neuropathy is Charcot-Marie-Tooth disorder, Guillain-Barre syndrome (acute inflammatory demyelinating polyradiculopathy type), schwannomatosis, chronic inflammatory demyelinating polyneuropathy, nerve trauma, post-chemotherapy neuropathy, diabetic peripheral neuropathy, migraine, Schwannomas, neurofibromatosis type 1 (NF1), malignant peripheral nerve sheath tumors (MPNSTs), endometriosis abdominopelvic pain or nerve injury.
  • Charcot-Marie-Tooth disorder Guillain-Barre syndrome (acute inflammatory demyelinating polyradiculopathy type), schwannomatosis, chronic inflammatory demyelinating polyneuropathy, nerve trauma, post-chemotherapy neuropathy, diabetic peripheral neuropathy, migraine, Schwannomas, neurofibromatosis type 1 (NF1), malignant peripheral nerve sheath tumors (MPNSTs), endometriosis abdomino
  • the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to reduce expression of the disease-associated molecule by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.
  • the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to reduce activity of the disease-associated molecule by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.
  • the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to reduce activity of the disease-associated molecule by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.
  • the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to reduce the expression of PMP22 by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some instances, the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to reduce the expression of PMP22 by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.
  • the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to reduce the activity of PMP22 by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some instances, the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to reduce the activity of PMP22 by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.
  • the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to reduce the expression of antiganglioside antibodies by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some instances, the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to reduce the expression of antiganglioside antibodies by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.
  • the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to reduce the activity of antiganglioside antibodies by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some instances, the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to reduce the activity of antiganglioside antibodies by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.
  • the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to increase expression of the disease-associated molecule by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.
  • the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to increase expression of the disease-associated molecule by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.
  • the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to increase activity of the disease-associated molecule by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.
  • the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to increase activity of the disease-associated molecule by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.
  • the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to increase the expression of the subunit of SWI/SNF protein complexes encoded by gene SMARCB1 by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.
  • the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to increase the expression of the subunit of SWI/SNF protein complexes encoded by gene SMARCB1 by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.
  • the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to reduce the activity of the subunit of SWI/SNF protein complexes encoded by gene SMARCB1 by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.
  • the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to reduce the activity of the subunit of SWI/SNF protein complexes encoded by gene SMARCB1 by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.
  • the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to increase the expression of LZTR1 protein encoded by gene LZTR1 by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some instances, the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to increase the expression ofLZTRl protein encoded by gene LZTR1 by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.
  • the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to reduce the activity ofLZTRl protein encoded by gene LZTR1 by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. In some instances, the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to reduce the activity ofLZTRl protein encoded by gene LZTR1 by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.
  • the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to increase expression of a marker gene of myelination by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.
  • Schwann cells exist in different developmental stages or wound repair phases, which express different Schwann cell-specific markers.
  • the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to increase expression of a marker gene of Schwann cell precursors by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.
  • the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to increase expression of a marker gene of immature Schwann cell by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.
  • the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to increase expression of a marker gene of myelinating Schwann cell by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.
  • the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to increase expression of a marker gene of non-myelinating Schwann cell by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.
  • the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to increase expression of one or more the markers, including but are not limited to, SI 00, p75 NTR , SoxlO, Sox2, GAP43, NCAM, Krox20, Oct6, MBP, and MPZ, at least by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.
  • the markers including but are not limited to, SI 00, p75 NTR , SoxlO, Sox2, GAP43, NCAM, Krox20, Oct6, MBP, and MPZ, at least by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.
  • kits for treating or preventing Neurofibroma and associated indications, disorders or types comprising: providing the drug conjugates disclosed herein or the pharmaceutical compositions disclosed herein; and administering to the subject the drug conjugates or the pharmaceutical compositions to treat the Neurofibroma and associated indications, disorders or types, to alleviate or reduce symptoms of the Neurofibroma and/or associated indications, disorders or types.
  • the Neurofibroma disease- associated molecule may be selected from receptor tyrosine kinases (RTKs), for example, of the Epidermal growth factor receptor family B (ErbB), such as EGFR, ErbB2, and ErbB3; additional RTKs include platelet-derived growth factor (PDGF), fibroblasts growth factor (FGF), insulin-like growth factor- 1 and vascular endothelial growth factor (VEGF).
  • RTKs receptor tyrosine kinases
  • ErbB Epidermal growth factor receptor family B
  • additional RTKs include platelet-derived growth factor (PDGF), fibroblasts growth factor (FGF), insulin-like growth factor- 1 and vascular endothelial growth factor (VEGF).
  • nrPTKs include Src, BCR-ABL, and the JAK/STAT pathway.
  • a drug conjugate or a pharmaceutical composition as disclosed herein comprises SMW compound as a drug molecule inhibiting kinase domain of RTKs or nrPTKs (e.g. exemplified above) as potential therapeutic pathway to neurofibromas.
  • the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to reduce expression of the disease-associated molecule by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.
  • the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to reduce expression of the disease-associated molecule by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.
  • the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to reduce activity of the disease-associated molecule by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.
  • the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to reduce activity of the disease-associated molecule by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.
  • the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to reduce the expression of RTKs or nrPTKs (e.g. exemplified above) by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.
  • the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to reduce the expression of RTKs or nrPTKs (e.g.
  • the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to reduce the activity of RTKs or nrPTKs (e.g. exemplified above) by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.
  • the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to reduce the activity of RTKs or nrPTKs (e.g. exemplified above) by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.
  • the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to reduce the activity of RTKs or nrPTKs (e.g. exemplified above) by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, [0225]
  • the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to increase expression of the disease-associated molecule by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.
  • the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to increase expression of the disease-associated molecule by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.
  • the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to increase activity of the disease-associated molecule by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.
  • the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to increase activity of the disease-associated molecule by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.
  • the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to increase the expression of the subunit of RTKs or nrPTKs (e.g. exemplified above) by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.
  • the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to increase the expression of RTKs or nrPTKs (e.g.
  • the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to increase the activity of RTKs or nrPTKs (e.g. exemplified above) by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.
  • the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to increase the activity of RTKs or nrPTKs (e.g. exemplified above) by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.
  • the drug conjugate or the pharmaceutical composition is administered in a dose and schedule effective to increase the activity of RTKs or nrPTKs (e.g. exemplified above) by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.
  • a dose and schedule effective to increase the activity of RTKs or nrPTKs (e.g. exemplified above) by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.
  • kits for treating or diagnosing a subject with a disease associated with dysfunction and/or degeneration of Schwann cells and/or axons, and axon related disorders comprising administering to a subject in need of such a treatment or diagnosis an effective amount of the drug conjugate as disclosed herein or a pharmaceutical composition as disclosed herein, comprising the drug conjugate.
  • drug conjugate encompasses, in a non-limiting manner, Antibody Drug Conjugate (ADC), Antibody Oligonucleotide Conjugate (AOC), Degraderantibody conjugates (DAC) or any conjugation of a therapeutic entity to a delivery entity.
  • ADC Antibody Drug Conjugate
  • AOC Antibody Oligonucleotide Conjugate
  • DAC Degraderantibody conjugates
  • disease-associated molecule generally refers to a molecule that is directly or indirectly associated with the pathogenesis, progression, prolonging, or recurrence of a disease.
  • the abnormal expression level of the disease-associated molecule is associated with the pathogenesis, progression, prolonging, or recurrence of the disease.
  • the abnormal activity of the disease-associated molecule is associated with the pathogenesis, progression, prolonging, or recurrence of the disease.
  • protein degraders or “degraders” as used herein refers to small molecular weight compounds that use the cell’s natural system (ubiquitin-or ubiquitin-like proteasome machinery) for disposing of spent or misfolded proteins. These small molecules are designed with two active ends: one that binds to the protein of interest and the other that binds to a ubiquitin ligase (protein called E3). These bifunctional molecules force a contact between the ubiquitin and the protein, applying it to be degraded. Afterwords, the process is repeated, enabling quick depletion of the unwanted protein. Thus the degraders are tagging the undesirable protein for ubiquitination and subsequent breakdown into amino acids in the proteasome.
  • DACs Degrader-antibody conjugates
  • PROTAC proteolysis targeting chimera
  • molecule payload an antibody (e.g. monoclonal antibody), usually via a type of chemical linker.
  • conservative amino acid substitution generally refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made.
  • Variants can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art such as are found in references which compile such methods, e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Fourth Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2012, or Current Protocols in Molecular Biology, F. M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York.
  • amino acids include substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.
  • an antibody generally refers to a polypeptide that includes at least one immunoglobulin variable domain or at least one antigenic determinant, e.g., paratope that specifically binds to an antigen.
  • an antibody is a full-length antibody.
  • an antibody is a chimeric antibody.
  • an antibody is a humanized antibody.
  • an antibody is a Fab fragment, a F(ab')2 fragment, a Fv fragment or an scFv fragment.
  • Fab fragment refers to a region on an antibody that binds to antigens. It is composed of one constant and one variable domain of each of the heavy and the light chain. These domains shape the paratope, which is the antigen-binding site, at the amino terminal end of the monomer.
  • Fc region refers to the tail region of an antibody that interacts with cell surface receptors called Fc receptors and some proteins of the complement system. This property allows antibodies to activate the immune system.
  • Fc regions of immunoglobulins bear a highly conserved N-glycosylation site.
  • an antibody is a nanobody derived from a camelid antibody or a nanobody derived from shark antibody.
  • an antibody is a diabody.
  • an antibody comprises a framework having a human germline sequence.
  • an antibody comprises a heavy chain constant domain selected from the group consisting of IgG, IgGl, IgG2, IgG2A, IgG2B, IgG2C, IgG3, IgG4, IgAl, IgA2, IgD, IgM, and IgE constant domains.
  • an antibody comprises a heavy (H) chain variable region (abbreviated herein as VH), and/or a light (L) chain variable region (abbreviated herein as VL).
  • VH heavy chain variable region
  • VL light chain variable region
  • an antibody comprises a constant domain, e.g., an Fc region.
  • An immunoglobulin constant domain refers to a heavy or light chain constant domain. Human IgG heavy chain and light chain constant domain amino acid sequences and their functional variations are known.
  • the heavy chain of an antibody described herein can be an alpha (a), delta (A), epsilon (s), gamma (y) or mu (p) heavy chain.
  • the heavy chain of an antibody described herein can comprise a human alpha (a), delta (A), epsilon (s), gamma (y) or mu (p) heavy chain.
  • an antibody described herein comprises a human gamma 1 CHI, CH2, and/or CH3 domain.
  • the amino acid sequence of the VH domain comprises the amino acid sequence of a human gamma (y) heavy chain constant region, such as any known in the art.
  • Non-limiting examples of human constant region sequences have been described in the art, e.g., see U.S. Pat. No. 5,693,780 and Kabat E A et al., (1991) supra.
  • 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.
  • an antibody is modified, e.g., modified via glycosylation, phosphorylation, sumoylation, and/or methylation.
  • an antibody is a glycosylated antibody, which is conjugated to one or more sugar or carbohydrate molecules.
  • the one or more sugar or carbohydrate molecule are conjugated to the antibody via N-glycosylation, O-glycosylation, C- glycosylation, glypiation (GPI anchor attachment), and/or phosphoglycosylation.
  • the one or more sugar or carbohydrate molecule are monosaccharides, disaccharides, oligosaccharides, or glycans. In some instances, the one or more sugar or carbohydrate molecule is a branched oligosaccharide or a branched glycan. In some instances, the one or more sugar or carbohydrate molecule includes a mannose unit, a glucose unit, an N-acetylglucosamine unit, an N-acetylgalactosamine unit, a galactose unit, a fucose unit, or a phospholipid unit.
  • an antibody is a construct that comprises a polypeptide comprising one or more antigen binding fragments of the disclosure linked to a linker polypeptide or an immunoglobulin constant domain.
  • Linker polypeptides comprise two or more amino acid residues joined by peptide bonds and are used to link one or more antigen binding portions. Examples of linker polypeptides have been reported (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2: 1121-1123).
  • an antibody may be part of a larger immunoadhesion molecule, formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides.
  • immunoadhesion molecules include use of the streptavidin core region to make a tetrameric scFv molecule (Kipriyanov, S. M., et al. (1995) Human Antibodies and Hybridomas 6:93-101) and use of a cysteine residue, a marker peptide and a C-terminal polyhistidine tag to make bivalent and biotinylated scFv molecules (Kipriyanov, S. M., et al. (1994) Mol. Immunol. 31: 1047-1058).
  • covalent generally refers to a characteristic of two or more molecules being linked together via at least one covalent bond.
  • two molecules can be covalently linked together by a single bond, e.g., a disulfide bond or disulfide bridge, that serves as a linker between the molecules.
  • two or more molecules can be covalently linked together via a molecule that serves as a linker that joins the two or more molecules together through multiple covalent bonds.
  • a linker may be a cleavable linker.
  • a linker may be a non-cleavable linker.
  • the term “specifically binds,” as used herein, generally refers to the ability of a molecule to bind to a binding partner with a degree of affinity or avidity that enables the molecule to be used to distinguish the binding partner from an appropriate control in a binding assay or other binding context.
  • the term, “specifically binds”, refers to the ability of the antibody to bind to a specific antigen with a degree of affinity or avidity, compared with an appropriate reference antigen or antigens, that enables the antibody to be used to distinguish the specific antigen from others, e.g., to an extent that permits preferential targeting Schwann cells, through binding to the antigen, as described herein.
  • an antibody specifically binds to a target if the antibody has a KD for binding the target of 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 M, or less.
  • the terms “subject,” “individual,” “mammal”, and “patient,” as used herein, used interchangeably and generally refer to may be and refer to humans, as well as non-human mammals (e.g., non-human primates, canines, equines, felines, porcines, bovines, ungulates, lagomorphs, and the like).
  • the subject can be a human (e.g., adult male, adult female, adolescent male, adolescent female, male child, female child) under the care of a physician or other health worker in a hospital, as an outpatient, or other clinical context. In certain instances, the subject may not be under the care or prescription of a physician or other health worker.
  • pcDNA3.4-mouse anti-Gliomedin-mlgGl (mouse Immunoglobulin Gl) long or heavy chain (VH): amino acid sequence as set forth in SEQ ID NO:1 (within the scope are sequences having at least 90%, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 1) or a functional variant thereof.
  • pcDNA3.4-mouse anti-Gliomedin-mCk short or light chain amino acid sequence as set forth in SEQ ID NO: 2 (within the scope are sequences having at least 90%, such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:2) or a functional variant thereof.
  • host cells are plated in 6- well plate and virus supernatant with definite MOI are added. Cells are incubated at 37°C in a CO2 incubator until it is ready to assay for gene expression after 48-72 hours. Infection can be performed once, or multiple times, depending on the expression level detection.
  • the cells cultured with appropriate antibiotics concentration for about 2 weeks are collected for sorting.
  • single cell seeding model is selected and total 3 plates of cells in 96-well plates are seeded to try to achieve the density of 1 cell per well.
  • Cells grow under antibiotic selection for 2 ⁇ 3 weeks until single clones can be identified in some of the wells.
  • Cells growing up will be expanded into 24-well plate and then 6-well plate to get enough cells for screening by FACS or Western blot.
  • Validation assay will be performed to confirm the positive characterization before cry opreservation.
  • the cells are harvested according to standard procedures. The cells are resuspended with FACS buffer and 4% Formaldehyde to finally 2% Formaldehyde and incubated for 20 min at room temperature (RT). The cells are washed 2-3 times with FACS buffer. Then the cells are resuspended in permeabilization buffer (0.2% TritonX-100) and incubated for 5 min, RT. The cells are washed 2-3 times with FACS buffer and incubated with diluted primary antibody for desired time on ice. The cells are washed 2-3 times with FACS buffer. Then the cells are centrifugated and the supernatant is discarded before and after each wash. The cells are incubated with diluted secondary antibody for desired time on ice in the dark. Finally, the cells are resuspended in FACS buffer for FACS analysis.
  • Anti-Gliomedin Ab was purified to homogeneity as was confirmed by SDS-PAGE, shown by Fig.lA, and SEC (Superdex200, 60ml, XK16/30) shown by Fig. IB.
  • the invention encompasses a polynucleotide molecule comprising the nucleic acid sequence of any one of SEQ ID NOs: 25-29 or a fragment thereof, wherein the sequence or fragment exhibits at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% sequence identity to any one of SEQ ID NOs: 25-29.
  • RNAimax was made as follows: 18 pL of RNAimax +300 pL of opti-MEM for each condition.
  • Two control siRNAs included (Scrambled and ON-TARGETplus SMARTpool human KIF11).
  • siRNAs were prepared in Opti-MEM: 6 pL of 10 pM siRNA stock +300 pL of opti-MEM. The siRNA was combined with the diluted RNAimax and incubated at RT for 15 min. An amount of 150 pL/well mix was added per well. This leads to 1.5ml final volume and a 10 nM siRNA final RNA concentration in each well. The mixture was incubated overnight and was replaced with pre-warmed culture media. Viability (Beckman Vi-cell counter or CTG assay) was checked and 72h samples were collected.
  • C3-PMP mice (Also Known As: PMP22-C3; B6.Cg- Tg(PMP22)C3Fbas/J, The Jackson Laboratories) were intravenously injected with AntibodysiRNA-Conjugate (AOC) (as described above, e.g. Examples 1-3) or Antibody-scrambled- siRNA-Conjugate control variant.
  • AOC AntibodysiRNA-Conjugate
  • a target or a disease-associated molecule e.g.
  • Purified anti-Gliomedin was concentrated in PBS at a concentration of 1-2 mg/mL, later partially reduced with DTT at a final concentration of 10 mM. The solution was incubated at 37°C for 30 minutes followed by incubation with iodoacetamide to the at a final concentration of 20 mM for 30 minutes in the dark.
  • the activated anti-Gliomedin was purified from excess reagents by dialysis.
  • a solution of MMAE in buffer at a concentration of 1-2 mM solution was prepared which was incubated to the linker solution (maleimide- activated VC-PAB) and incubated at room temperature for 30 minutes.
  • Example 7 Evaluation of targeted delivery of an antibody-MMAE drug molecule conjugate (ADC) in vitro

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Abstract

Des conjugués de médicament comprenant une fraction de ciblage conjuguée à une molécule de médicament sont divulgués, la fraction de ciblage se liant à une fraction d'adhésion cellulaire et/ou un récepteur exprimé sur une cellule de Schwann pour induire une administration ciblée du conjugué de médicament à la cellule de Schwann, et la molécule de médicament modifiant l'expression ou l'activité d'une molécule associée à une maladie, et/ou conférant un effet cytotoxique, dans la cellule de Schwann. Des méthodes d'administration ciblée d'un médicament aux cellules de Schwann sont également divulguées.
PCT/IL2025/050035 2024-01-12 2025-01-12 Administration ciblée de conjugués de médicament à des cellules de schwann et méthodes de traitement dans des maladies associées à des cellules de schwann Pending WO2025150052A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN120860184A (zh) * 2025-07-23 2025-10-31 贵州医科大学 Cadm3在制备治疗自身免疫性疾病的产品中的应用

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WO2007000768A2 (fr) * 2005-06-28 2007-01-04 Yeda Research And Development Co. Ltd. At The Weizmann Institute Of Science Gliomedine: fragments et procedes
WO2008142693A2 (fr) * 2007-05-22 2008-11-27 Yeda Research And Development Co. Ltd. Régulation de la myélination par des molécules de type nectine (necl)
CN111514312A (zh) * 2020-05-22 2020-08-11 南通大学 一种促进神经再生多肽药物结合物及其应用
WO2021184021A1 (fr) * 2020-03-13 2021-09-16 Codiak Biosciences, Inc. Constructions de vésicules extracellulaires - aso ciblant pmp22
WO2021258011A1 (fr) * 2020-06-19 2021-12-23 Ionis Pharmaceuticals, Inc. Composés et procédés pour moduler pmp22
WO2022119826A1 (fr) * 2020-12-01 2022-06-09 Research Institute At Nationwide Children's Hospital Compositions et procédés pour l'inhibition de l'expression de la protéine 22 de la myéline périphérique
WO2022140521A1 (fr) * 2020-12-22 2022-06-30 Wisconsin Alumni Research Foundation Éléments régulateurs pour l'expression génique spécifique de cellules de schwann
WO2023091985A1 (fr) * 2021-11-18 2023-05-25 Dtx Pharma, Inc. Composés ciblant pmp22 pour le traitement de la maladie de charcot-marie-tooth
WO2024013563A1 (fr) * 2022-07-12 2024-01-18 Potentia Therapeutics Ltd Administration ciblée dans les cellules de schwann et méthodes de traitement dans des maladies associées aux cellules de schwann

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007000768A2 (fr) * 2005-06-28 2007-01-04 Yeda Research And Development Co. Ltd. At The Weizmann Institute Of Science Gliomedine: fragments et procedes
WO2008142693A2 (fr) * 2007-05-22 2008-11-27 Yeda Research And Development Co. Ltd. Régulation de la myélination par des molécules de type nectine (necl)
WO2021184021A1 (fr) * 2020-03-13 2021-09-16 Codiak Biosciences, Inc. Constructions de vésicules extracellulaires - aso ciblant pmp22
CN111514312A (zh) * 2020-05-22 2020-08-11 南通大学 一种促进神经再生多肽药物结合物及其应用
WO2021258011A1 (fr) * 2020-06-19 2021-12-23 Ionis Pharmaceuticals, Inc. Composés et procédés pour moduler pmp22
WO2022119826A1 (fr) * 2020-12-01 2022-06-09 Research Institute At Nationwide Children's Hospital Compositions et procédés pour l'inhibition de l'expression de la protéine 22 de la myéline périphérique
WO2022140521A1 (fr) * 2020-12-22 2022-06-30 Wisconsin Alumni Research Foundation Éléments régulateurs pour l'expression génique spécifique de cellules de schwann
WO2023091985A1 (fr) * 2021-11-18 2023-05-25 Dtx Pharma, Inc. Composés ciblant pmp22 pour le traitement de la maladie de charcot-marie-tooth
WO2024013563A1 (fr) * 2022-07-12 2024-01-18 Potentia Therapeutics Ltd Administration ciblée dans les cellules de schwann et méthodes de traitement dans des maladies associées aux cellules de schwann

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN120860184A (zh) * 2025-07-23 2025-10-31 贵州医科大学 Cadm3在制备治疗自身免疫性疾病的产品中的应用

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