EP4680642A2 - Nanobody-based protein degraders and related methods - Google Patents

Nanobody-based protein degraders and related methods

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Publication number
EP4680642A2
EP4680642A2 EP24771725.9A EP24771725A EP4680642A2 EP 4680642 A2 EP4680642 A2 EP 4680642A2 EP 24771725 A EP24771725 A EP 24771725A EP 4680642 A2 EP4680642 A2 EP 4680642A2
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EP
European Patent Office
Prior art keywords
seq
disease
protein
cpp
peptide
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP24771725.9A
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German (de)
French (fr)
Inventor
Nam CHU
Nhat LE
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Ohio State Innovation Foundation
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Ohio State Innovation Foundation
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22

Definitions

  • compositions comprising and/or encoding nanodegraders and methods of use thereof.
  • Protein folding diseases including Alzheimer's and Parkinson’s diseases, amyotrophic lateral sclerosis (ALS), prion diseases, and among others affect millions of people in the US.
  • ALS amyotrophic lateral sclerosis
  • prion diseases and among others affect millions of people in the US.
  • One of the earliest steps in the triggers of these diseases is the misfolding, aggregation, and accumulation of proteins in the central nervous system (CNS).
  • CNS central nervous system
  • compositions, vectors, and kits comprising a chimeric nanobody protein degrader.
  • the present disclosure provides methods of using compositions, vectors, and kits comprising a chimeric nanobody protein degrader.
  • the present disclosure also provides methods of treating or preventing a disease in a subject in need thereof.
  • the present disclosure also provides methods of decreasing or reducing a disease-related protein.
  • ND nanodegrader
  • Nb chimeric nanobody
  • PEC proximity-enabled crosslinker
  • the Nb comprises less than 200 amino acids or is less than 15kDa. In some embodiments, the Nb does not comprise a lysine residue. In some embodiments, the Nb comprises at least one protease-resistant D-amino acid.
  • the Nb comprises the Nb comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, or a variant thereof.
  • the CPP comprises a cyclic CPP or a membrane translocation domain (MTD). In some embodiments, the CPP comprises an arginine-rich cyclic CPP. In some embodiments, the CPP comprises CPP12, or a variant thereof. In some embodiments, the CPP comprises SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, or a variant thereof.
  • the targeting peptide comprises a chaperon-mediated autophagy (CMA) peptide, a macrocyclic (MC) peptide, E3 ligase ligand, or derivatives thereof.
  • the PEC comprises a proximity-enabled sulfur-fluoride exchange (SuFEx) crosslinker.
  • the Nb is irreversibly bound to a protein of interest (POI) by the PEC.
  • POI includes, but is not limited to a misfolded protein or a cancer- associated protein selected from tau, TAR DNA-binding protein 43 (TDP-43), a-synucleic, P- amyloid, prion proteins, human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR), CKLF like MARVEL transmembrane domain-containing 6 (CMTM6), human transcription factor cellular MYC (c-MYC), proliferating cell nuclear antigen (PCNA), and hypoxia-inducible factor 1-alpha (HIF-la).
  • HER2 human epidermal growth factor receptor 2
  • EGFR epidermal growth factor receptor
  • CKLF like MARVEL transmembrane domain-containing 6
  • c-MYC human transcription factor cellular MYC
  • PCNA proliferating cell nuclear antigen
  • HIF-la hypoxia-in
  • the pharmaceutically acceptable carrier comprises an excipient, a diluent, a salt, a buffer, a stabilizer, a lipid, an emulsion, or a native Nb.
  • the ND further comprises one or more protein labeling compounds.
  • the protein labeling compound comprises biotin, a reporter enzyme, a fluorophore, or a radioactive isotope.
  • a vector comprising a nucleic acid sequence encoding the ND, or a fragment thereof, of any preceding aspect.
  • kits comprising the ND, the vector, or a salt thereof, of any preceding aspect.
  • the kit further comprises one or more reagents for stabilizing the ND.
  • the kit further comprises one or more reagents for delivering the ND to a biological sample or subject.
  • the kit further comprises one or more reagents for amplifying the vector.
  • the kit further comprises one or more reagents for expressing the vector in a cell.
  • a method of treating or preventing a disease in a subject in need thereof comprises administering a pharmaceutically effective amount of a nanodegrader (ND) composition comprising a chimeric nanobody (Nb) of any preceding aspect and a pharmaceutically acceptable carrier, wherein the Nb is operably linked to a cell penetrating peptide (CPP), a targeting peptide, a proximity-enabled crosslinker (PEC), and wherein the ND targets and degrades a disease-related protein.
  • ND nanodegrader
  • Nb chimeric nanobody
  • PEC proximity-enabled crosslinker
  • ND nanodegrader
  • the method decreases or reduces the disease-related protein relative to an untreated control.
  • the disease-related protein comprises tau, TAR DNA-binding protein 43 (TDP-43), a-synucleic, P-amyloid, a prion protein, human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR), CKLF like MARVEL transmembrane domain-containing 6 (CMTM6), human transcription factor cellular MYC (c- MYC), proliferating cell nuclear antigen (PCNA), or hypoxia-inducible factor 1-alpha (HIF-la).
  • the method further comprises administering the ND and a therapeutic agent.
  • the therapeutic agent comprises an antibiotic, an antiinflammatory compound, a sedative, an anesthetic, an anti-viral agent, a peptide hormone, an antidiabetic agent, a steroid, or combinations thereof.
  • the disease comprises a neurodegenerative disease, a cancer, including but not limited to Alzheimer’s disease, ataxia, Huntington’s disease, Parkinson’s disease, amyotrophic lateral sclerosis (ALS), Friedreich ataxia, Lewy body disease, a prion disease, Creutzfeldt-Jakob disease, neurological cancers (including, but not limited to brain cancers), lung cancers, breast cancers, and gastric cancers.
  • a neurodegenerative disease a cancer, including but not limited to Alzheimer’s disease, ataxia, Huntington’s disease, Parkinson’s disease, amyotrophic lateral sclerosis (ALS), Friedreich ataxia, Lewy body disease, a prion disease, Creutzfeldt-Jakob disease, neurological cancers (including, but not limited to brain cancers), lung cancers, breast cancers, and gastric cancers.
  • a method of degrading or eliminating a disease-related protein in a subject or biological sample comprising introducing a nanodegrader (ND) composition to the subject or biological sample, wherein the ND comprises a chimeric nanobody (Nb) of any preceding aspect and pharmaceutically acceptable carrier, wherein the Nb is operably linked to a cell penetrating peptide (CPP), a targeting peptide, and a proximity-enabled crosslinker (PEC), internalizing the ND into a cell of the subject or biological sample, wherein the CPP directs internalization into the cell, irreversibly targeting and binding the ND to the disease-related protein, wherein the PEC binds the disease-related protein to the ND, and degrading the disease- related protein, wherein the targeting peptide activates at least one proteolytic pathway to decrease the disease-related protein relative to a control subject or biological sample.
  • ND nanodegrader
  • the ND comprises a chimeric nanobody (Nb) of any preceding aspect and
  • a method of degrading or eliminating a disease-related protein in a subject or biological sample comprising introducing a nanodegrader (ND) composition of any preceding aspect to the subject or biological sample, internalizing the ND into a cell of the subject or biological sample, wherein the CPP directs internalization into the cell, irreversibly targeting and binding the ND to the disease-related protein, wherein the PEC binds the disease-related protein to the ND, and degrading the disease-related protein, wherein the targeting peptide activates at least one proteolytic pathway to decrease the disease-related protein relative to a control subject or biological sample.
  • ND nanodegrader
  • the method of any preceding aspect comprises a Nb comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, or a variant thereof.
  • the method of any preceding aspect comprises a ND comprising a CPP with SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, or a variant thereof.
  • the PEC comprises a proximity-enabled sulfur-fluoride exchange (SuFEx) crosslinker.
  • the Nb is irreversibly bound to the disease-related protein of any preceding aspect by the PEC.
  • the pharmaceutically acceptable carrier comprises an excipient, a diluent, a salt, a buffer, a stabilizer, a lipid, an emulsion, or a native Nb.
  • the method of any preceding aspect comprises one or more protein labeling compounds of any preceding aspect.
  • the at least one proteolytic pathway comprises a ubiquitin- proteosome pathway, a lysosomal proteolytic pathway, or a combination thereof.
  • the method treats or prevents a disease selected from Alzheimer’s disease, ataxia, Huntington’s disease, Parkinson’s disease, amyotrophic lateral sclerosis (ALS), Friedreich ataxia, Lewy body disease, a prion disease, or Creutzfeldt-Jakob disease.
  • a disease selected from Alzheimer’s disease, ataxia, Huntington’s disease, Parkinson’s disease, amyotrophic lateral sclerosis (ALS), Friedreich ataxia, Lewy body disease, a prion disease, or Creutzfeldt-Jakob disease.
  • the biological sample comprises a tissue biopsy, a cerebrospinal fluid (CSF) sample, a blood sample, a serum sample, or an isolated cell.
  • CSF cerebrospinal fluid
  • Figures 1A, IB, and 1C show a platform technology.
  • Figure 1A shows that targeting misfolded proteins or cancer-associated proteins are developed and applied on specialized human neuronal models of diseases of Figure IB.
  • Figure 1A shows a diagram of nanobodybased protein molecules, including nano-degrader (ND) and nano-crosslinker (NC).
  • Nanobody (Nb) is attached with CPP (cell-penetrating peptide), targeting sequences (such as CMA, chaperon mediated autophagy, MC peptides) and chemical species (such as TMR, Tetramethyl- rhodamine fluorophore), biotin and proximity-enabled covalent crosslinkers.
  • CPP cell-penetrating peptide
  • targeting sequences such as CMA, chaperon mediated autophagy, MC peptides
  • chemical species such as TMR, Tetramethyl- rhodamine fluorophore
  • Figure IB shows a two-layer human iPSC-derived neuronal culture system.
  • Functioning human neurons (Figure B2), with a representative result of single- neuron patch-clamp recordings ( Figure Bl), are cultured on coverslips suspended over a feeder layer of glial cells ( Figure B3, microglia).
  • Immunofluorescence imaging of mature neurons and microglia staining for cell type markers including Tuj 1 (Class III B-Tubulin), Vglutl (Vesicular glutamate transporter 1), and Ibal (Ionized calcium-binding adaptor molecule 1).
  • Figure 1C shows the Nb on the ND/NCs recognizes and covalently crosslinks with targets (disease-associated proteins) at the membrane and/or in the cytoplasm via cross- linker fluorosulfonate amino acids (FS-AAs).
  • targets disease-associated proteins
  • FS-AAs cross- linker fluorosulfonate amino acids
  • Figures 2A, 2B, 2C, and 2D show the protein pathology in human disease neuronal models with mutation, (Figure 2A) ALS- TARDBP Q331K , ( Figure 2B) ALS/FTD-UBQLN2 P497H , ( Figure 2C) PD-SNCA A53T and ( Figure 2D) CJD-PRNP E200K .
  • Immunoassay shows the disease hallmark accumulation of pathogenic proteins in human neuronal cultures with mutations, including misallocated TDP43 ( Figures 2A and 2B), ubiquilin2 (Figure 2B), a-syn ( Figure 2C), prion protein (Figure 2D), and pathological Tau (PHF Tau).
  • Mutant neurons with TARDBP Q331K , SCNA A53T and UBQLN P497H/P506T are derived from iisogenic IPSCs.
  • the neurons with PRNP F200K are derived from patient IPSCs.
  • PK proteinase K treatment.
  • ScN2a a prion-infected cell line derived from N2a.
  • SMI-31 neuro-filament maker.
  • D2 shows neurofibrillary tau tangles-like pathology in PRNP E200K neurons.
  • Figures 3A and 3B show the mutant neurons display a lower number of dendritic spines compared to normal neurons.
  • Figures 3A shows that the cultures were stained with fluorescent phalloidin (red) for F-actin. Spines were visualized and quantified.
  • Figure 3B shows that the pooled measurements of staining were collected from 30-40 regions from 2-3 replicated cultures.
  • Figures 4A, 4B, 4C, 4D, 4E, and 4F show the development of NCs.
  • Figure 4A shows a schematic for the proximity-enabled cross-linking of Nb with POI-protein of interest using SuFex chemistry with fluorosulfate amino acids FS-Tyrosine (FSY) and FS-Lysine (K-FS) incorporated into Nb.
  • Figure 4B shows that NC ALFA was treated on ALFA-eGFP- expressed live neurons.
  • IP Immunoprecipitation
  • Figure 4C shows a schematic for ND with the attachments of CPP, CMA and chemical species, including TMR fluorophore.
  • Figure 4D shows the SDS gel for the conjugation of semisynthetic CMA-Nb ALFA with TMR.
  • Figure 4E shows the SDS gel for conjugating CMA- Nb ALFA -TMR with CPP peptide.
  • Figure 4F shows the duel function NDs strategies.
  • Figures 5 A, 5B, 5C, 5D, 5E, 5F, 5G, 5H, and 51 show the successful degradation of ALFA- eGFP by ND ALFA in Hela cells ( Figures 5A, 5B, and 5C) and neurons ( Figures 5D, 5E, and 5F).
  • Blots Figure 5B
  • eGFP and ND ALFA were detected by anti-HA and -biotin antibodies, respectively.
  • IF images of eGFP-expressed neurons exposed to Nb ALFA Figure 5D and E).
  • ND ALFA was decorated with TMR (red), and biotin. Arrows indicated co-localization of ND and eGFP in the cells. Quantifications show a significant decrease in eGFP level in ND-treated cell ( Figure 5C) and neuron (Figure 5F) samples. Data is from pool measurements of 3 different cultures. NDs target a-syn ( Figures 5G, 5H, and 51), ND a -syn2. C] ⁇ jA-NbSyn2-CPP-TMR.
  • FIG. 5H shows the cellular internalization and a-syn degradation of NDs“' syn2 (red, TMR) in Hela cells expressed a-syn proteins (green) (aggregate A53T a-syn protein indicated by arrows). Arrow heads show co-localization of ND“' syn2 molecules and a-syn proteins.
  • Figure 51 shows the quantifications of a-syn intensity shows reducing of signal of ND treated samples compare to non-treated samples.
  • Figures 6A, 6B, 6C, and 6D show the studying of PrP interactomes.
  • Figure 6A shows the SDS gel for the purification of PrP nanobody (Nb PrP ) and conjugation of Nb PrP with CPP.
  • Figure 6C and 6D shows the interaction of Nb PrP -based proteins (shown in panel F i gure 6 A), including Nb PrP ( Figure 6C) and Nb PrP -CPP ( Figure 6D), with PrP in Hela.
  • Hela cells overexpressing PrP ( Figure 6C) or both PrP and UBQLN2 P497H ( Figure 6D) were treat with Nb PrP or Nb PrP -CPP for 2 hours.
  • Nb PrP - based proteins green
  • C2 membrane PrP
  • DI cytoplasmic PrP
  • NC PrP strategy with live ineurons at membrane (C2) and cytoplasm (D2).
  • Protein mapping with UV light and diazirine-Nb PrP are used to generate reactive carbenes that cross-link nearby proteins within 1 nm radius (green area). Selecting labeling occurs through immunoreaction of photocatalyst-NC PrP .
  • Figure 6B shows the photo-proximity protein work flow on live neurons.
  • Figures 7A, 7B, 7C, and 7D show the development of nano-degrader.
  • Figure 7A shows the bifunctional nano-degrader brings together El, E2, E3 ligase complex and POI resulting in proteasomal degradation of POI.
  • Figure 7B shows the schematic for the generation of nanodegrader contains E3 ligase ligand, POI-specific nanobody using EPL, followed by the attachment of cell-permeable peptide (CPP).
  • Figure 7C shows the SDS-PAGE analysis for EPL of recombinant Nb(l-97)-Intein fusion without (lane 1, control) and with the synthetic Nb(98-122) peptide (lane 2).
  • Figure 7D shows the pulldown assay for GST-ALFA _Nb fusion proteins: wild type (WT-Nb, left) and non-Lysine variant (KR-Nb, right) immobilized on glutathione beads and then mixed with ALFA-tagged sortase A (ALFA-SrtA). The beads were washed and then analyzed by SDS-gel.
  • Figures 8A and 8B show the differentiation of human iPSCs into neurons and microglial cells.
  • An example of misfolded Tau protein in prion diseases has been characterized in iPSC- derived cell models. Accumulation of pathological Tau (green), the paired helical filaments (PHF- Tau) aggregated forms (white arrows), in human IPSC-derived neurons from patients carrying E200K mutation of prion protein, which causes Creutzfeldt-Jakob Disease (a familial prion disease in human), as compared to the non-carrier (NC).
  • Figures 9A, 9B and 9C show the level of total a-synuclein (a-syn) protein after the treatment with ND21 on human neuron contain A53T mutation.
  • Figure 9A shows the schematic for the generation of nanodegrader 21 (ND21) contains CMA1 peptide, syn 2 nanobody using EPL with malamide (Ma) conjugation, followed by the attachment of cell-permeable peptide (CPP) CR10.
  • Figures 9B and 9C show IPSCs- derived human neuron with A53T a-syn mutation at day 28 of differentiation, which were used to test the ND21.
  • Figure 9B shows the Western Blot results of neurons were treated with ND21 at 0.5uM final concentration for 32 and 96 hours (A53T + ND21). Neurons in the same batch were treated with same amount of PBS buffer as a control (A53T). Wild-type neurons at the same day-old were used as a control. After the treatments, neuron cultures were collected for Western blotting. The proteins were detected by Western blot analysis using antibodies shown.
  • Figure 9C show graphical data of total asyn protein level in the lysate samples in this experiment. Data shown is the mean ⁇ SEM.
  • Figures 10A, 10B and IOC show the internalization and a-syn degradation effects of the ND21 in the soma region of human neurons.
  • IPSCs- derived human neuron with A53T asyn mutation at day 28 of differentiation were used to test the ND21.
  • Figure 10B shows neurons were treated with ND21 at 0.5uM final concentration for 32 hours (A53T + ND21).
  • Figure 10A shows neurons in the same batch were treated with same amount of PBS buffer as control (A53T).
  • Figures 10 A and B show neuron cultures after these treatments, which were fixed and stained for a-syn (green) and HA tag (red).
  • ND21 treated a-syn A53T neurons show reduction of a-syn protein level compared to the non-treated samples.
  • ND21 which is positive to HA staining in treated neurons, is located in the soma regions (Bl).
  • the boxed regions in each panel are shown at higher magnification in the smaller horizontal panels to the right (Al A2 and Bl, B2).
  • Figure 10C shows the quantitation of cytoplasmic a-syn integrated density levels show the fold change of a-syn in the soma regions of these neurons. Pooled measurements were collected from 15-20 neurons from 3 different cultures. Data shown is the mean ⁇ SEM.
  • Figures 11A, 1 IB and 11C show the rescue effects of ND21 on synaptotoxicity of human neurons with a-syn A53T mutation.
  • IPSCs- derived human neuron with A53T asyn mutation at day 28 of differentiation were used to test the ND21.
  • Figure 1 IB shows neurons were treated with ND21 at 0.5uM final concentration for 32 hours (A53T + ND21).
  • Figure 11A shows neurons in the same batch were treated with same amount of PBS buffer as control (A53T).
  • Figures 11 A and 1 IB show neuron cultures after the treatments which were fixed and stained with Phalloidin (red) forF-Actinto visualize the dendritic spine morphologies.
  • ND21 treated asyn A53T neurons shown increasing number of spines compare to the non-treated samples.
  • the boxed regions (Al, Bl) in each panel are shown at higher magnification in the smaller horizontal panels to the right (Al, Bl).
  • Figure 11C shows the quantitation of spine number shows number of dendritic spines per lOpM. Pooled measurements were collected from dendri sections of 15-20 neurons from 3 different cultures. Data shown is the mean ⁇ SEM.
  • Figures 12A, 12B, 12C show the schematic illustration for using copper-catalyzed azidealkyne cycloaddition (Cu-AAC) to incorporate proximity-enabled crosslinker fluorosulfonate (FSY) amino acids into Nanobodies (Nbs).
  • Cu-AAC copper-catalyzed azidealkyne cycloaddition
  • FSY proximity-enabled crosslinker fluorosulfonate
  • Nbs Nanobodies
  • the FSY can be incorporated into the Lysine side chain directly or via a variable PEG(n>2) linker.
  • Figures 13A, 13B, 13C, and 13D show schematic illustrations for genetic code expansion.
  • Figure 13 A shows the schematic illustration for genetic code expansion with the amber codon (TAG) suppression to incorporate 4-Azido Phenylalanine (Azido-Phe) into Nanobodies (Nbs).
  • TAG amber codon
  • Azido-Phe 4-Azido Phenylalanine
  • Nbs Nanobodies
  • aaRS aminoacyl-tRNA-synthetase
  • Azido-Phe magenta star
  • FIG. 13B shows the copper-free azdie-alkyne cycloaddition for Alfa-tag Nb containing Azido-Phe with dibenzocyclooctyne (DBCO) sulfo-Cy5 (fluorophore probe, Lumiprobe). Left panel is for coomassie staining and right panel is for Cy5 fluorescence scanning at 700 nm wavelength. M: protein marker.
  • Figure 13C shows the peptide carrier (5) for use with click chemistry to incorporate E3 ligase ligands into Nbs.
  • Figure 13D show the copper-free azide-DBCO cycloaddition for Azido-Phe-Nb with E3 -ligand-linked DBCO- peptide.
  • Figures 14A, 14B, and 14C show illustrations of ND delivery and degradation mechanism.
  • Figure 14A illustrates the NDs cellular delivery and degradation mechanisms.
  • Figure 14B shows the SDS-PAGE analysis of CMA1 developed HER2-ND conjugated with CPP.
  • Figure 14C shows the immunofluorescent image confirming the colocalization of Nb-CMAl construct and HER2 in SKBR3 cells.
  • Figure 15 shows that vector map of the pET-22b-PelB-TEV-HA-Nanobody-3C-6xHis plasmid.
  • Figure 16 shows the vector map of the pET28-TEV-Cys-HA-Nanobody-3C-6xHis plasmid.
  • Figure 17 shows the vector map of the pTXBl-TEV-C-HA-Nanobody-CMAl plasmid.
  • Figures 18 A, 18B, 18C, 18D, and 18E show the various nanodegrader constructs.
  • Figure 18A shows the ectodomain-targeting nanobody.
  • Figure 18B shows the covalent binding nanodegrader (ND).
  • Figure 18C shows the cytoplasmic-targeting nanobody.
  • Figure 18D shows the biparatopic ND.
  • Figure 18E shows the proteasome-targeting ND.
  • FIG. 19 shows the cell-penetrating peptides (CPPS) used herein.
  • the CPPs of the present disclosure include Di-TAMRA-TAT peptide comprising SEQ ID NO: 34, or a variant thereof, Cys-cyclic-TAT peptide comprising SEQ ID NO: 35, or a variant thereof, or Cys-cyclic- Deca-Arginine peptide comprising SEQ ID NO: 36, or a variant thereof.
  • Figure 20 shows the peptide (SEQ ID NO: 2), or a variant thereof, used for incorporation of proximity-enabled crosslinker fluorosulfonate (FSY) into nanobodies using protein semi synthesis strategy.
  • Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed.
  • composition refers to any agent that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition.
  • composition also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, a vector, polynucleotide, cells, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like.
  • composition includes the composition per se as well as pharmaceutically acceptable, pharmacologically active vector, polynucleotide, salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc.
  • compositions, methods, etc. include the recited elements, but do not exclude others.
  • Consisting essentially of' when used to define compositions and methods shall mean including the recited elements, but excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like.
  • Consisting of' shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions provided and/or claimed in this disclosure. Embodiments defined by each of these transition terms are within the scope of this disclosure.
  • An “increase” can refer to any change that results in a greater amount of a symptom, disease, composition, condition, or activity.
  • An increase can be any individual, median, or average increase in a condition, symptom, activity, composition in a statistically significant amount.
  • the increase can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% increase so long as the increase is statistically significant.
  • a “decrease” can refer to any change that results in a smaller amount of a symptom, disease, composition, condition, or activity.
  • a substance is also understood to decrease the genetic output of a gene when the genetic output of the gene product with the substance is less relative to the output of the gene product without the substance.
  • a decrease can be a change in the symptoms of a disorder such that the symptoms are less than previously observed.
  • a decrease can be any individual, median, or average decrease in a condition, symptom, activity, composition in a statistically significant amount.
  • the decrease can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% decrease so long as the decrease is statistically significant.
  • reduce or other forms of the word, such as “reducing” or “reduction,” means lowering of an event or characteristic (e.g., misfolded protein aggregation). It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to.
  • reduced protein misfolding means reducing the amount of misfolded protein relative to a standard or a control.
  • prevent or other forms of the word, such as “preventing” or “prevention,” is meant to stop a particular event or characteristic, to stabilize or delay the development or progression of a particular event or characteristic, or to minimize the chances that a particular event or characteristic will occur. Prevent does not require comparison to a control as it is typically more absolute than, for example, reduce. As used herein, something could be reduced but not prevented, but something that is reduced could also be prevented. Likewise, something could be prevented but not reduced, but something that is prevented could also be reduced. It is understood that where reduce or prevent are used, unless specifically indicated otherwise, the use of the other word is also expressly disclosed.
  • treat include partially or completely delaying, alleviating, mitigating or reducing the intensity of one or more attendant symptoms of a disorder or condition and/or alleviating, mitigating or impeding one or more causes of a disorder or condition.
  • Treatments according to the disclosure may be applied preventively, prophylactically, palliatively or remedially. Treatments are administered to a subject prior to onset (e.g., before obvious signs of neurodegeneration), during early onset (e.g., upon initial signs and symptoms of neurodegeneration), or after an established development of neurodegenerati on .
  • the term “subject” refers to any individual who is the target of administration or treatment.
  • the subject can be a vertebrate, for example, a mammal.
  • the subject can be human, non-human primate, bovine, equine, porcine, canine, or feline.
  • the subject can also be a guinea pig, rat, hamster, rabbit, mouse, or mole.
  • the subject can be a human or veterinary patient.
  • patient refers to a subject under the treatment of a clinician, e.g., physician.
  • amino acid includes but is not limited to amino acids contained in the group consisting of alanine (Ala or A), cysteine (Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Gly or G), histidine (His or H), isoleucine (He or I), lysine (Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (Gin or Q), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine (Vai or V), tryptophan (Trp or W), and tyrosine (Tyr or Y) residues.
  • amino acid residue also may include amino acid residues contained in the group consisting of homocysteine, 2- Aminoadipic acid, N-Ethylasparagine, 3 -Aminoadipic acid, Hydroxylysine, P-alanine, P- Aminopropionic acid, allo-Hydroxylysine acid, 2- Aminobutyric acid, 3-Hydroxyproline, 4- Aminobutyric acid, 4-Hydroxyproline, piperidinic acid, 6-Aminocaproic acid, Isodesmosine, 2- Aminoheptanoic acid, allo-Isoleucine, 2-Aminoisobutyric acid, N-Methylglycine, sarcosine, 3- Aminoisobutyric acid, N-Methylisoleucine, 2-Aminopimelic acid, 6-N-Methyllysine, 2,4- Diaminobutyric acid, N-Meth
  • a polypeptide and/or protein is defined as a polymer of amino acids, typically of length>100 amino acids (Garrett & Grisham, Biochemistry, 2nd edition, 1999, Brooks/Cole, 110).
  • a peptide is defined as a short polymer of amino acids, of a length typically of 20 or less amino acids, and more typically of a length of 12 or less amino acids (Garrett & Grisham, Biochemistry, 2nd edition, 1999, Brooks/Cole, 110).
  • peptides, polypeptides, and proteins disclosed herein may be modified to include nonamino acid moieties. Modifications may include but are not limited to carboxylation (e.g., N- terminal carboxylation via addition of a di-carboxylic acid having 4-7 straight-chain or branched carbon atoms, such as glutaric acid, succinic acid, adipic acid, and 4,4-dimethylglutaric acid), amidation (e.g., C-terminal amidation via addition of an amide or substituted amide such as alkylamide or dialkylamide), PEGylation (e.g., N-terminal or C-terminal PEGylation via additional of polyethylene glycol), acylation (e.g., O-acylation (esters), N-acylation (amides), S- acylation (thioesters)), acetylation (e.g., the addition of an acetyl group, either at the N-terminus of the protein or at lysine
  • glycation Distinct from glycation, which is regarded as a nonenzymatic attachment of sugars, polysialylation (e.g., the addition of poly sialic acid), glypiation (e.g., glycosylphosphatidylinositol (GPI) anchor formation, hydroxylation, iodination (e.g., of thyroid hormones), and phosphorylation (e.g., the addition of a phosphate group, usually to serine, tyrosine, threonine, or histidine).
  • polysialylation e.g., the addition of poly sialic acid
  • glypiation e.g., glycosylphosphatidylinositol (GPI) anchor formation
  • hydroxylation e.g., hydroxylation
  • iodination e.g., of thyroid hormones
  • phosphorylation e.g., the addition of a phosphate
  • amino acids, and derivatives occurs in two isomeric forms: L-forms or D-forms.
  • L- and D- forms represent the same atoms of an amino acid, however the atoms can have different arrangements, which can impact the amino acid properties and functions.
  • the two forms are similar in that they both occur naturally and comprise a central carbon atom, at least one hydrogen atom, a carboxylic group, an amine group, and a variable group.
  • the two forms differ in that they are usually mirrored images of each other, wherein the location of the amine group varies.
  • L-amino acids are used in protein synthesis, while D-amino acid are less common in protein synthesis.
  • L-amino acids rotate counterclockwise or to the left in a process known as levorotation.
  • D-amino acids rotate clockwise or to the right in a process known as dextrorotation.
  • a “fusion protein” refers to a protein formed by the fusion of at least one peptide, polypeptide, protein or variant thereof as disclosed herein to at least one molecule of a heterologous peptide, polypeptide, protein or variant thereof.
  • the heterologous protein(s) may be fused at the N-terminus, the C-terminus, or both termini.
  • a fusion protein comprises at least a fragment or variant of the heterologous protein(s) that are fused with one another, preferably by genetic fusion (i.e., the fusion protein is generated by translation of a nucleic acid in which a polynucleotide encoding all or a portion of a first heterologous protein is joined in-frame with a polynucleotide encoding all or a portion of a second heterologous protein).
  • the heterologous protein(s), once part of the fusion protein may each be referred to herein as a “portion”, “region” or “moiety” of the fusion protein.
  • variant means a polypeptide derived from a parent polypeptide by one or more (several) alteration(s), i.e., a substitution, insertion, and/or deletion, at one or more (several) positions.
  • a substitution means a replacement of an amino acid occupying a position with a different amino acid;
  • a deletion means removal of an amino acid occupying a position;
  • an insertion means adding 1 or more, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably 1-3 amino acids immediately adjacent an amino acid occupying a position.
  • ‘immediately adjacent’ may be to the N-side (‘'upstream’) or C-side (‘downstream’ ) of the amino acid occupying a position (‘the named amino acid'). Therefore, for an amino acid named/numbered ‘X,’ the insertion may be at position ‘ X+T (‘downstream’) or at position ‘X--1 ’ (‘upstream').
  • a “variant” of a particular polypeptide sequence may be defined as a polypeptide sequence having at least 50% sequence identity to the particular polypeptide sequence over a certain length of one of the polypeptide sequences using blastp with the “BLAST 2 Sequences” tool available at the National Center for Biotechnology Information's website. (See Tatiana A. Tatusova, Thomas L. Madden (1999), “Blast 2 sequences — a new tool for comparing protein and nucleotide sequences”, FEMS Microbiol Lett. 174:247-250).
  • a variant polypeptide may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to a reference polypeptide.
  • a variant polypeptide may have substantially the same functional activity as a reference polypeptide.
  • a variant polypeptide may exhibit or more biological activities associated with binding a ligand and/or binding DNA at a specific binding site.
  • a “nucleotide” is a compound consisting of a nucleoside, which consists of a nitrogenous base and a 5-carbon sugar, linked to a phosphate group forming the basic structural unit of nucleic acids, such as DNA or RNA.
  • the four types of nucleotides are adenine (A), cytosine (C), guanine (G), and thymine (T), each of which are bound together by a phosphodiester bond to form a nucleic acid molecule.
  • nucleic acid is a chemical compound that serves as the primary information-carrying molecules in cells and make up the cellular genetic material.
  • Nucleic acids comprise nucleotides, which are the monomers made of a 5-carbon sugar (usually ribose or deoxyribose), a phosphate group, and a nitrogenous base.
  • a nucleic acid can also be a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA).
  • a chimeric nucleic acid comprises two or more of the same kind of nucleic acid fused together to form one compound comprising genetic material.
  • a “full length” polynucleotide sequence is one containing at least a translation initiation codon (e.g., methionine) followed by an open reading frame and a translation termination codon.
  • a “full length” polynucleotide sequence encodes a “full length” polypeptide sequence.
  • a variant polynucleotide may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to a reference polynucleotide.
  • vector refers to any vehicle that carries a polynucleotide into a cell for the expression of the polynucleotide in the cell.
  • the vector may be, for example, a plasmid, a virus, a phage particle, or a nanoparticle. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may in some instances, integrate into the genome itself.
  • the vector is a DNA construct containing a DNA sequence which is operably linked to a suitable control sequence capable of effecting the expression of the DNA in a suitable host cell.
  • control sequences can include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites, and sequences which control the termination of transcription and translation.
  • a plasmid or a viral vector can be capable of extrachromosomal replication or, optionally, can integrate into the host genome.
  • the term "integrated" used in reference to an expression vector means the expression vector, or a portion thereof, is incorporated (physically inserted or ligated) into the chromosomal DNA of a host cell.
  • a “viral vector” refers to a virus-like particle containing genetic material which can be introduced into a eukaryotic cell without causing substantial pathogenic effects to the eukaryotic cell.
  • viruses or viral vectors can be used for transduction but should be compatible with the cell type the virus or viral vector are transduced into (e.g., low toxicity, capability to enter cells).
  • Suitable viruses and viral vectors include adenovirus, lentivirus, retrovirus, among others.
  • the expression vector encoding a chimeric polypeptide is a naked DNA or is comprised in a nanoparticle (e.g., liposomal vesicle, porous silicon nanoparticle, gold-DNA conjugate particle, polyethyleneimine polymer particle, cationic peptides, etc.).
  • the vector is a lipid nanoparticle. Lipid nanoparticles can be used to deliver mRNA to a host cell for expression of the mRNA in the host cell.
  • administer refers to delivering a composition, substance, inhibitor, or medication to a subject or object by one or more the following routes: oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-joint, parenteral, intra-arteriole, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation or via an implanted reservoir.
  • parenteral includes subcutaneous, intravenous, intramuscular, intraarticular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injections or infusion techniques.
  • kit describes a wide variety of bags, containers, carrying cases, and other portable enclosures which may be used to carry and store solid substances, liquid substances, and other accessories necessary to stabilize, administering, store, and/or express the compositions and vectors disclosed herein.
  • “Pharmaceutically acceptable carrier” (sometimes referred to as a “carrier”) means a carrier or excipient that is useful in preparing a pharmaceutical or therapeutic composition that is generally safe and non-toxic, and includes a carrier that is acceptable for veterinary and/or human pharmaceutical or therapeutic use.
  • carrier or “pharmaceutically acceptable carrier” can include, but are not limited to, phosphate buffered saline solution, water, emulsions (such as an oil/water or water/oil emulsion) and/or various types of wetting agents.
  • carrier encompasses any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations.
  • a carrier for use in a composition will depend upon the intended route of administration for the composition.
  • the preparation of pharmaceutically acceptable carriers and formulations containing these materials is described in, e.g., Remington's Pharmaceutical Sciences, 21st Edition, ed. University of the Sciences in Philadelphia, Lippincott, Williams & Wilkins, Philadelphia, PA, 2005.
  • physiologically acceptable carriers include saline, glycerol, DMSO, buffers such as phosphate buffers, citrate buffer, and buffers with other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN (ICI, Inc.; Bridgewater, New Jersey), polyethylene glycol (PEG), and PLURONICS (BASF; Florham Park, NJ).
  • buffers such as phosphate buffers,
  • TPD targeted protein degradation
  • ND nanobody-based protein degraders
  • the PROTAC method employs the ubiquitin-proteasome system (UPS), a major intracellular protein degradation system, to degrade a protein of interest (POI).
  • UPS ubiquitin-proteasome system
  • POI protein of interest
  • this method has been only applied to targets with available small molecule inhibitors, and classically “undruggable” proteins remain challenging. Particularly, this strategy also proves unworkable for targeting misfolded proteins in neurodegenerative diseases.
  • therapeutic compositions comprising NDs are developed and tested herein for targeting pathogenic proteins toward intracellular degradation processes.
  • compositions, vectors, and kits comprising a chimeric nanobody protein degrader.
  • present disclosure provides methods of using compositions, vectors, and kits comprising a chimeric nanobody protein degrader.
  • the present disclosure provides therapeutic molecules targeting the degradation of pathological proteins.
  • the Targeted Protein Degradation (TPD) system is called nanobody-based protein degraders (nano-degraders, NDs).
  • NDs nanobody-based protein degraders
  • CPP conjugated cell-penetrating peptide
  • CMA chaperon-mediated autophagy
  • small E3 ligase selective ligands allow rapid degradation of targeted protein in lysosomes and/or proteasome.
  • the resultant NDs will be employed to target pathological proteins in protein misfolding diseases and cancers.
  • the present disclosure also provides disease-associated neuronal culture models derived from human IPSCs, which successfully recapitulate the diseases with several key pathological features, including the accumulation of pathological proteins, to assess the rescue efficiency of the NDs in a realm of human context.
  • ND molecules are synthesized and characterized that target cellular pathological a-syn proteins and 2) it is demonstrated that ND degrades pathological a-syn and moderates the neurotoxic effects in the human PD-a-syn neuronal cultures.
  • ND nanodegrader
  • Nb chimeric nanobody
  • PEC proximity-enabled crosslinker
  • Nanobodies are currently the smallest antibody molecules with a molecular weight of 1/10 of a normal antibody.
  • nano-antibodies possess unique functional properties, such as small molecular mass, strong stability, good solubility, easy expression, weak immunogenicity, strong penetrability, strong targeting, simple in humanization, low in preparation cost, etc.
  • Nb use often improves the shortcomings of traditional antibody long-term development cycle, low stability, and harsh storage conditions.
  • the antigen-binding properties of a nanobody can be described by three specific regions located in the variable region of the heavy chain, referred as variable regions (CDRs), and the segment is divided into four frame regions (FRs).
  • CDRs variable regions
  • FRs frame regions
  • the amino acid sequences of four FRs are conservative and do not directly participate in binding reactions. These CDRs form a loop structure in which the P-sheets formed by the FRs therebetween are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen- binding site of the nanobody.
  • the amino acid sequences of the same type of nanobodies can be compared to determine which amino acids constitute the FR or CDR regions.
  • variable regions of the heavy chains of the nanobodies of the invention become a particular interest because at least a part of them is involved in binding antigens.
  • the present invention includes those molecules having a nanobody heavy chain variable region with a CDR, provided that their CDRs are 90% or more (preferably 95% or more, the most preferably 98% or more) identical to the CDRs identified herein.
  • the present disclosure includes not only intact nanobodies but also fragment(s) of immunologically active nanobody or fusion protein(s) formed from nanobodies with other sequences. Therefore, the present disclosure also includes fragments, derivatives, and analogs of the nanobodies.
  • fragment refers to a polypeptide that substantially retains the same biological function or activity of a nanobody of the invention.
  • Polypeptide fragments, derivatives or analogs of the invention may be (i) polypeptides having one or more conservative or non-conservative amino acid residues (preferably non-conservative amino acid residues) substituted.
  • Such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) a polypeptide having a substituent group in one or more amino acid residues; or (iii) a polypeptide formed by fusing a mature polypeptide and another compound (such as a compound that increases the half-life of the polypeptide, for example, polyethylene glycol); or (iv) a polypeptide formed by fusing an additional amino acid sequence to the polypeptide sequence (e.g., a leader or secretory sequence or a sequence used to purify this polypeptide or a proprotein sequence, or a fusion protein formed with a 6 His tag).
  • these fragments, derivatives, and analogs are within the scope of one of ordinary skill in the art.
  • the nanobody of the present disclosure refers to a polypeptide including the CDR regions.
  • the term also encompasses variant forms of polypeptides comprising the above CDR regions that have the same function as the nanobodies of the invention. These variations include, but are not limited to, deletion insertions and/or substitutions of one or several (usually 1-50, preferably 1-30, more preferably 1-20, optimally 1-10) amino acids, and addition of one or several (generally less than 20, preferably less than 10, and more preferably less than 5) amino acids at C-terminus and/or N-terminus.
  • the substitution of amino acids with analogic or similar properties usually does not alter the function of the protein.
  • addition of one or several amino acids at the C-terminus and/or N-terminus usually does not change the function of the protein.
  • the term also includes active fragments and active derivatives of the nanobodies of the invention.
  • variant forms of the polypeptide include homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNAs capable of hybridizing with DNA encoding the nanobody of the present invention under high or low stringent conditions, and polypeptides or proteins obtained using antiserum against the nanobodies of the invention.
  • the invention also provides other polypeptides, such as a fusion protein comprising nanobodies or fragments thereof.
  • the present invention also includes fragments of the nanobodies of the invention.
  • the fragment has at least about 50 contiguous amino acids of the nanobody of the invention, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids.
  • a conservative variant of a nanobody of the present invention refers to the polypeptides in which there are up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids substituted by amino acids having analogical or similar properties, compared to the amino acid sequence of the nanobody of the present invention.
  • the nanobodies herein may be used alone or in combination or conjugated with a detectable marker (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modification moiety, or a combination thereof.
  • a detectable marker for diagnostic purposes
  • a therapeutic agent for therapeutic purposes
  • a PK (protein kinase) modification moiety for therapeutic purposes
  • a PK protein kinase
  • the Nb comprises less than 200 amino acids or is less than 15kDa. In some embodiments, the Nb comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
  • the Nb is 0.5kDa, 1 kDa, 2 kDa, 3kDa, 4kDa, 5kDa, 6kDa, 7kDa, 8kDa, 9kDa, lOkDa, l lkDa, 12kDa, 13kDa, 14kDa, 15kDa.
  • Natural L-amino acids serve as the starting components for protein synthesis and protein- related research. D-amino acids share identical chemical and physical properties with L-amino acids, however D-amino acids rotate plane polarized light in the opposite direction relative to L- amino acids. In regards to protein-related research, peptides are increasingly becoming attractive drug candidate to treat various diseases. However, it has become recognized that peptides containing L-amino acids a highly susceptible to protein degradation. It has become recognized that peptides containing D-amino acids are more resistant to endogenous protein degradation. Thus, the present disclosure provides an ND composition comprising peptides with at least one D-amino acid, wherein the D-amino acids prevent protein degradation of the components of the ND. In some embodiments, the Nb comprises at least one protease-resistant D-amino acid.
  • the Nb comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, or a variant thereof.
  • the CPP comprises a cyclic CPP or a membrane translocation domain (MTD). In some embodiments, the CPP comprises an arginine-rich cyclic CPP. In some embodiments, the CPP comprises CPP12, or a variant thereof. In some embodiments, the CPP comprises SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, or a variant thereof.
  • the CPP comprises the constructs including but not limited to Di- TAMRA-TAT peptide comprising SEQ ID NO: 34, or a variant thereof; Cys-cyclic-TAT peptide comprising SEQ ID NO: 35, or a variant thereof; or Cys-cyclic-Deca-Arginine peptide comprising SEQ ID NO: 36, or a variant thereof.
  • the CPP comprises any one of the CPP of Figure 19, or a variant thereof.
  • the ubiquitin-proteosome pathway is a selective protein degradation pathway that uses ubiquitin (a 76 amino acid polypeptide to mark proteins for degradation. Proteins are marked for degradation by the attachment of ubiquitin to lysine residues.
  • ubiquitin a 76 amino acid polypeptide
  • Proteins are marked for degradation by the attachment of ubiquitin to lysine residues.
  • the present disclosure provides ND compositions having minimal or completely eliminating lysine residues.
  • the Nb does not comprise a lysine residue.
  • the NDs herein may optionally have ubiquitin-recruiting ligase (E3 ligase, E3 ubiquitin ligase) binding moiety (binder).
  • the ubiquitin ligase binder may be an analog of thalidomide, which binds the E3 ubiquinase known as cereblon.
  • the ubiquitin ligase binder may also be a ligand that binds the von Hippel-Lindau tumor suppressor (VHL) protein, which is attached via a linker to another small molecule (target- moiety) that binds a target protein (see, e.g., Lai et cd.. Angew. Chem. Int. Ed.
  • Targeted protein degradation refers to small molecule (e.g. , protein binding small molecule) induced ubiquitination and degradation of disease targets, in which the small molecule may simultaneously recruit both a ubiquitin ligase and the target protein into proximity of each other, which may lead to ubiquitination of the target protein.
  • small molecule e.g. , protein binding small molecule
  • the small molecule may simultaneously recruit both a ubiquitin ligase and the target protein into proximity of each other, which may lead to ubiquitination of the target protein.
  • Protein degradation is a highly regulated and essential process that maintains cellular homeostasis.
  • the selective identification and removal of damaged, misfolded, or excess proteins is achieved via the ubiquitin-proteasome pathway (UPP).
  • UPP ubiquitin-proteasome pathway
  • the UPP in fact is central to the regulation of almost all cellular processes, including antigen processing, apoptosis, biogenesis of organelles, cell cycling, DNA transcription and repair, differentiation and development, immune response and inflammation, neural and muscular degeneration, morphogenesis of neural networks, modulation of cell surface receptors, ion channels and the secretory pathway, the response to stress and extracellular modulators, ribosome biogenesis and viral infection.
  • Covalent attachment of multiple ubiquitin molecules by an E3 ubiquitin ligase to a terminal lysine residue marks the protein for proteasome degradation, where the protein is digested into small peptides and eventually into its constituent amino acids that serve as building blocks for new proteins.
  • Defective proteasomal degradation has been linked to a variety of clinical disorders including Alzheimer's disease, Parkinson's disease, Huntington's disease, muscular dystrophies, cardiovascular disease, and cancer among others.
  • the targeting peptide comprises a chaperon-mediated autophagy (CMA) peptide, a macrocyclic (MC) peptide, E3 ligase ligand, or derivatives thereof.
  • the targeting peptide comprises SEQ ID NO: 9, or a variant thereof.
  • the targeting peptide comprises SEQ ID NO: 33, or a variant thereof.
  • the targeting peptide comprises an amino acid sequence of a chaperon-mediated autophagy (CMA) peptide, a macrocyclic (MC) peptide, E3 ligase ligand, or derivatives thereof.
  • the PEC comprises a proximity-enabled sulfur-fluoride exchange (SuFEx) crosslinker.
  • the Nb is irreversibly bound to a protein of interest (POI) by the PEC.
  • the POI comprises a misfolded protein selected from tau, TAR DNA- binding protein 43 (TDP-43), a-synuclein, P-amyloid, or prion proteins.
  • the POI comprises an accumulated oncoprotein or a cancer-associated protein including, but not limited to human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR), CKLF like MARVEL transmembrane domain-containing 6 (CMTM6), human transcription factor cellular MYC (c-MYC), proliferating cell nuclear antigen (PCNA), and hypoxia-inducible factor 1 -alpha (HIF-la), immunosuppressive protein, cytokines, or chemokines including, but not limited to transforming growth factor-beta (TGF-P) and interleukin- 10 (IL-10), or variants thereof.
  • HER2 human epidermal growth factor receptor 2
  • EGFR epidermal growth factor receptor
  • c-MYC human transcription factor cellular MYC
  • PCNA proliferating cell nuclear antigen
  • HIF-la hypoxia-inducible factor 1 -alpha
  • the pharmaceutically acceptable carrier comprises an excipient, a diluent, a salt, a buffer, a stabilizer, a lipid, an emulsion, or a native Nb.
  • the ND further comprises one or more protein labeling compounds.
  • the protein labeling compound comprises biotin, a reporter enzyme, a fluorophore, or a radioactive isotope.
  • the fluorophore includes, but are not limited to, 1,5 IAEDANS; 1,8- ANS; 4- Methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein; 5-Carboxyfluorescein (5- FAM); 5-Carboxynapthofluorescein; 5-Carboxytetramethylrhodamine (5-TAMRA); 5-Hydroxy Tryptamine (5-HAT); 5-ROX (carboxy-X-rhodamine); 6-Carboxyrhodamine 6G; 6-CR 6G; 6- JOE; 7-Amino-4-methylcoumarin; 7- Aminoactinomycin D (7-AAD); 7-Hydroxy-4- I methylcoumarin; 9-Amino-6-chloro-2-methoxyacridine (ACMA); ABQ; Acid Fuchsin; Acridine Orange; Acridine Red; Acridine Yellow; Acriflavin; Acriflavin
  • the reporter enzyme includes, but is not limited to luciferase, green fluorescent protein (GFP), yellow fluorescent protein (YFP), blue fluorescent protein (BFP), cyane fluorescent protein (CFP), monomeric red fluorescent protein (mRFP), Discosoma striata (DsRed), mCherry, mOrange, tdTomato, mSTrawberry, mPlum, photoactivatable GFP (PA-GFP), Venus, Kaede, monomeric kusabira orange (mKO), Dronpa, enhanced CFP (ECFP), Emerald, Cyan fluorescent protein for energy transfer (CyPet), super CFP (SCFP), Cerulean, photoswitchable CFP (PS-CFP2), photoactivatable RFP1 (PA-RFP 1), photoactivatable mCherry (PA-mCherry), monomeric teal fluorescent protein (mTFPl), Eos fluorescent protein (EosFP), Dendra, TagBFP, TagRFP, enhanced YFP
  • GFP
  • the radioactive isotope includes, but is not limited to technetium- 99m, caesium- 137, chromium-51, iodine- 123, palladium- 103, molybdenum-99, iodine-131, iridium- 192, lutetium- 177, copper-64, radium-226, cobalt-60, tritium, americium-241, samarium- 153, and yttrium-90.
  • the present disclosure also discloses an ND comprising the structure: A-B-C, wherein either A, B, or C comprises a component to direct proteosome degradation of the POI, a component to direct lysosome/autophagosome degradation of the POI, or a component that binds to the POI.
  • the component directing proteosome degradation comprises a ubiquitin ligand or a ubiquitin ligand complex.
  • ubiquitin ligand or ubiquitin ligand complex comprises Skpl-Cullin-F-box ubiquitin ligase complex, murine double minute 2 (MDM2) ubiquitin ligase, cereblon (CRBN), cell inhibitor of apoptosis protein (cIAP), and Von- Hipple-Lindau (VHL), including VHL as part of the CRL2VHL E3 ligase complex, complexed with Cullin 4A (CUL4A), DNA-binding protein 1 (DDB1), and ring box protein 1 (RBX1).
  • the ubiquitin ligand/ubiquitin ligand complex comprises an E3 ligand.
  • the component directing lysosome/autophagosome degradation comprises chaperone-mediated autophagy peptides/motifs or chaperone-mediated autophagy protein complex.
  • the chaperone-mediated peptide/protein or the chaperone- mediated peptide/protein complex comprises B-crystallin, p23, DJ-1, and heat shock protein chaperones, including, but not limited to Hsp90, Hsp70, Hsp27, and Hsp60.
  • the chaperone-mediated protein/chaperone-mediated protein complex comprises a heat shock protein (Hsp).
  • ubiquitin ligand/ubiquitin ligand complex and the chaperone- mediated protein/chaperone-mediated protein complex are linked by a linker.
  • the ND further comprises moieties including, but not limited to cationicindependent mannose-6-phosphate receptor (CI-MPR aka IGF2FR), poly-M6Pn, asialoglycoprotein receptor (ASGPR), and DNA aptamers Al and A2.
  • the ND of any preceding aspect comprises a construct necessary to achieve the desired effect, such as for example targeting the ectodomain, targeting the cytoplasm, and/or targeting the proteasome.
  • the ND of any preceding aspect comprises an ectodomain-targeting domain construct ( Figure 18 A), a covalent binding construct (Figure 18B), a cytoplasmic-targeting nanobody construct (Figure 18C), a bi-paratopic construct (Figure 18D), and/or a proteasome-targeting domain construct (Figure 18E).
  • the concerned sequences can be obtained in large scale using recombinant methods. Usually, sequences can be obtained by cloning it into a vector, transferring it into cells, and then isolating the sequences from the proliferated host cells by conventional methods.
  • Bio-molecules (nucleic acids, proteins, etc.) to which the present invention relates include bio-molecules that exist in isolated form.
  • DNA sequences encoding the protein of the present invention can be obtained completely by chemical synthesis.
  • the DNA sequence then can be introduced into various existing DNA molecules (or e.g., vectors) and cells known in the art.
  • mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
  • the vector comprising a nucleic acid sequence encoding the ND, or a fragment thereof, of any preceding aspect.
  • the vector is a plasmid, a virus or a viral vector, a phage particle, or a nanoparticle.
  • the vectors comprise the above-mentioned suitable DNA sequences and suitable promoters or control sequences. These vectors can be used to transform an appropriate host cell so that it can express the protein.
  • the vector includes, but is not limited to a plasmid derived from pTXBl, pET22, and pET28.
  • the vector comprises the plasmid of Figure 15.
  • the vector comprises the plasmid of Figure 16. In some embodiments, the vector comprises the plasmid of Figure 17. In some embodiments, the vector is suitable for integration and/or expression of a fusion protein, such as for example the ND comprising the Nb, the CPP, and the targeting peptide. In some embodiments, the vector comprises a PelB sequence suitable for localizing and/or expressing the ND in the periplasm.
  • the vector of any preceding aspect comprises one or more suitable control sequence capable of effecting the expression of the DNA in a suitable host cell.
  • suitable control sequences can include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites, and sequences which control the termination of transcription and translation.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a mammalian cell.
  • Representative examples are: Escherichia coli, Slreplomyces.
  • bacterial cells such as Salmonella typhimurium, fungal cells such as yeast, insect cells of Drosophila S2 or Sf9, animal cells of CHO, COST, 293 cells, and the like.
  • the transformation of the host cell with the recombinant DNA can be performed using conventional techniques well known to those skilled in the art.
  • competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with the CaC12 method.
  • the procedures used are well known in the art.
  • Another method is to use MgC12. If necessary, conversion can also be performed by electroporation.
  • DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, and the like.
  • the obtained transformants can be cultured in a conventional manner to express the polypeptide encoded by the gene of the present invention.
  • the medium used in the culture may be selected from various conventional media.
  • the culture is performed under conditions suitable for the host cells growth. After the host cells are grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature shift or chemical induction) and the cells are incubated for a further period of time.
  • the recombinant polypeptide in the above method may be expressed intracellularly, or on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods by utilizing its physical, chemical, and other characteristics. These methods are well-known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitation agent (salting out method), centrifugation, osmotic disruption, super treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer analysis, ion exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques and the combinations thereof.
  • conventional renaturation treatment treatment with a protein precipitation agent (salting out method), centrifugation, osmotic disruption, super treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer analysis, ion
  • kits comprising the ND, the vector, or a salt thereof, of any preceding aspect.
  • the kit further comprises one or more reagents for stabilizing the ND.
  • the kit further comprises one or more reagents for delivering the ND to a biological sample or subject.
  • the kit further comprises one or more reagents for amplifying the vector.
  • the kit further comprises one or more reagents for expressing the vector in a cell.
  • the kit further comprises instructions for using the ND, vector, or a salt thereof.
  • the kit further includes a container, an instruction, a buffer, and the like.
  • the present disclosure also provides disease-associated neuronal culture models comprising two-layers of co-culturing microglia and neurons derived from isogenic induced pluripotent stem cells (iPSC) lines.
  • iPSC isogenic induced pluripotent stem cells
  • the present disclosure also provides methods of treating or preventing a disease in a subject in need thereof.
  • the present disclosure also provides methods of decreasing or reducing a disease-related protein.
  • a nanodegrader and methods of use thereof can be applied to any disease affected by or associated with a pathogenic protein.
  • the present disclosure describes a method of administering the ND composition comprising a nanoody (Nb), wherein the ND targets a pathogenic protein.
  • pathogenic protein refers to a fragment or full-length peptide, polypeptide, protein that is produced as a result of a disease or disorder, or is defective or mutated causing the onset or progression of a disease or disorder.
  • the appearance of pathogenic proteins leads to worsening of disease symptoms, addition of more disease symptoms, activation of immunoprotective and/or immunosuppressive signaling pathways, and/or maybe asymptomatic.
  • pathogenic proteins can be endogenous to the subject or arise from an exogenous sources, including, but not limited to the environment, bacteria, virus, fungi, protozoa, and parasites.
  • the pathogenic proteins are expressed from genetic mutations or disruptions.
  • a method of treating or preventing a disease in a subject in need thereof comprises administering a pharmaceutically effective amount of an ND composition comprising a. Nb and a pharmaceutically acceptable carrier, wherein the Nb is operably linked to a cell penetrating peptide (CPP), a targeting peptide, a proximity-enabled crosslinker (PEC), and wherein the ND targets and degrades a disease-related protein, including but not limited to tau, TAR DNA-binding protein 43 (TDP-43), a-synuclein, P-amyloid, a prion protein, human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR), CKLF like MARVEL transmembrane domain-containing 6 (CMTM6), human transcription factor cellular MYC (c-MYC), proliferating cell nuclear antigen (PCNA), or hypoxia-inducible factor 1-alpha (HIF-la).
  • CPP cell penetrating peptide
  • ND nanodegrader
  • the method decreases or reduces the disease-related protein relative to an untreated control. In some embodiments, the methods decreases or reduces the disease- related protein by 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% relative to an untreated control.
  • the method further comprises administering the ND and a therapeutic agent.
  • the therapeutic agent comprises an antibiotic, an antiinflammatory compound, a sedative, an anesthetic, an anti-viral agent, a peptide hormone, an antidiabetic agent, a steroid, or combinations thereof.
  • the therapeutic agent includes, but is not limited to penicillins (including, but not limited to amoxicillin, clavulanate and amoxicillin, ampicillin, dicloxacillin, oxacillin, and penicillin V potassium), tetracyclins (including, but not limited to demeclocycline, doxycycline, eravacycline, minocycline, omadacycline, sarecycline, and tetracycline), cephalosporins (cefaclor, cefadroxil, cefdinir, cephalexin, cefprozil, cefepime, cefiderocol, cefotaxime, cefotetan, ceftaroline, cefazidme, ceftriaxone, and cefuroxime), quinolones (also referred to as fluoroquinolones include, but are not limited to ciprofloxacin, delafloxacin, levof
  • HERCEPTIN (trastuzumab), T-DM1, AVASTIN (bevacizumab), ERBITUX (cetuximab), VECTIBIX (panitumumab), RITUXAN (rituximab), BEXXAR (tositumomab)
  • anti-estrogens e.g. tamoxifen, raloxifene, and megestrol
  • LHRH agonists e.g. goscrclin and leuprolide
  • anti-androgens e.g. flutamide and bicalutamide
  • photodynamic therapies e.g.
  • BPD-MA vertoporfm
  • phthalocyanine phthalocyanine
  • photosensitizer Pc4 demethoxy-hypocrellin A
  • demethoxy-hypocrellin A demethoxy-hypocrellin A
  • nitrogen mustards e.g. cyclophosphamide, ifosfamide, trofosfamide, chlorambucil, estramustine, and melphalan
  • nitrosoureas e.g. carmustine (BCNU) and lomustine (CCNU)
  • alkyl sulphonates e.g. busulfan and treosulfan
  • triazenes e.g. dacarbazine, temozolomide
  • platinum containing compounds e.g.
  • paclitaxel or a paclitaxel equivalent such as nanoparticle albumin-bound paclitaxel (ABRAXANE), docosahexaenoic acid bound-paclitaxel (DHA-paclitaxel, Taxoprexin), polyglutamate bound- paclitaxel (PG-paclitaxel, paclitaxel poliglumex, CT-2103, XYOTAX), the tumor-activated prodrug (TAP) ANG1005 (Angiopep-2 bound to three molecules of paclitaxel), paclitaxel -EC- 1 (paclitaxel bound to the erbB2-recognizing peptide EC-1), and glucose-conjugated paclitaxel, e.g., 2'-paclitaxel methyl
  • etoposide etoposide phosphate, teniposide, topotecan, 9-aminocamptothecin, camptoirinotecan, irinotecan, crisnatol, mytomycin C
  • anti-metabolites DHFR inhibitors (e.g. methotrexate, dichloromethotrexate, trimetrexate, edatrexate), IMP dehydrogenase inhibitors (e.g. mycophenolic acid, tiazofurin, ribavirin, and EICAR), ribonucleotide reductase inhibitors (e.g. hydroxyurea and deferoxamine), uracil analogs (e.g.
  • 5 -fluorouracil 5-FU
  • floxuridine doxifluridine, ratitrexed, tegafur-uracil, capecitabine
  • cytosine analogs e.g. cytarabine (ara C), cytosine arabinoside, and fludarabine
  • purine analogs e.g. mercaptopurine and Thioguanine
  • Vitamin D3 analogs e.g. EB 1089, CB 1093, and KH 1060
  • isoprenylation inhibitors e.g. lovastatin
  • dopaminergic neurotoxins e.g. l-methyl-4-phenylpyridinium ion
  • cell cycle inhibitors e.g.
  • actinomycin e.g. actinomycin D, dactinomycin
  • bleomycin e.g. bleomycin A2, bleomycin B2, peplomycin
  • anthracycline e.g. daunorubicin, doxorubicin, pegylated liposomal doxorubicin, idarubicin, epirubicin, pirarubicin, zorubicin, mitoxantrone
  • MDR inhibitors e.g. verapamil
  • Ca 2+ ATPase inhibitors e.g.
  • thapsigargin imatinib, thalidomide, lenalidomide, tyrosine kinase inhibitors (e.g., axitinib (AG013736), bosutinib (SKI- 606), cediranib (RECENTINTM, AZD2171), dasatinib (SPRYCEL®, BMS-354825), erlotinib (TARCEVA®), gefitinib (IRESSA®), imatinib (Gleevec®, CGP57148B, STI-571), lapatinib (TYKERB®, TYVERB®), lestaurtinib (CEP-701), neratinib (HKI-272), nilotinib (TASIGNA®), semaxanib (semaxinib, SU5416), sunitinib (SUTENT®, SU11248), toceranib (PALLADIA®), vandetani
  • the disease comprises a neurodegenerative disease, an inflammatory disease, a viral infection, or a cancer.
  • the disease includes, but is not limited to Alzheimer’s disease, ataxia, Huntington’s disease, Parkinson’s disease, amyotrophic lateral sclerosis (ALS), Frontotemporal dementia (FTD), Friedreich ataxia, Lewy body disease, spinal muscular atrophy, Alpers’ disease, Batten disease, Cerebro-oculo-facio- skeletal syndrome, Leigh syndrome, Prion diseases, monomelic amyotrophy, multiple system atrophy, striatonigral degeneration, motor neuron disease, multiple sclerosis (MS), Creutzfeldt- Jakob disease, Parkinsonism, spinocerebellar ataxia, dementia, acoustic neuroma, adenocarcinoma, adrenal gland cancer, anal cancer, angiosarcoma (e.g., lymphangiosarcoma, lymphangioendotheliosarcoma,
  • HCC hepatocellular cancer
  • lung cancer e.g., bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung
  • myelofibrosis MF
  • chronic idiopathic myelofibrosis chronic myelocytic leukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilic syndrome (HES)
  • gliomas neuroblastoma, neurofibroma (e.g., neurofibromatosis (NF) type 1 or type 2, schwannomatosis), neuroendocrine cancer (e.g., gastroenteropancreatic neuroendoctrine tumor (GEP-NET), carcinoid tumor), osteosarcoma, ovarian cancer (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma), papillary adenocarcinoma, pancreatic cancer (e.g., pancreatic adenocarcinoma, intraductal papillary mucinous neoplasm (IPMN), Islet cell tumors), penile cancer (e.g., Page
  • cytochrome c oxidase (COX) deficiency LHON Leber Hereditary Optic Neuropathy; MM Mitochondrial Myopathy: LIMM Lethal Infantile Mitochondrial Myopathy; MMC Maternal Myopathy and Cardiomyopathy; NARP Neurogenic muscle weakness, Ataxia, and Retinitis Pigmentosa; Leigh Disease: FICP — Fatal Infantile Cardiomyopathy Plus, a MELAS-associated cardiomyopathy: MELAS Mitochondrial Encephalomyopathy with Lactic Acidosis and Stroke-like episodes; LDYT Leber's hereditary optic neuropathy and Dystonia; MERRF Myoclonic Epilepsy and
  • the ND composition may be administered in such amounts, time, and route deemed necessary in order to achieve the desired result.
  • the exact amount of the ND composition will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular ND composition, its mode of administration, its mode of activity, and the like.
  • the ND composition is preferably formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the ND composition will be decided by the attending physician within the scope of sound medical judgment.
  • the specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disease being treated and the severity of the disease symptoms; the activity of the ND composition employed; the specific ND composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific ND composition employed; the duration of the treatment; drugs used in combination or coincidental with the ND composition employed; and like factors well known in the medical arts.
  • the ND composition may be administered by any route.
  • the ND composition is administered via a variety of routes, including oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, mucosal, nasal, buccal, enteral, sublingual; by intratracheal instillation, bronchial instillation, and/or inhalation; and/or as an oral spray, nasal spray, and/or aerosol.
  • routes including oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, mucosal, nasal, buccal, enteral, sublingual; by intratracheal instillation, bronchial instillation, and/or inhalation; and/or as an oral spray, nasal spray, and/or aerosol.
  • the most appropriate route of administration will depend upon a variety of factors including the nature of the ND composition (e.g., its stability in the environment of the subject’s body), the condition of the subject (e.g., whether the subject is able to tolerate oral administration), etc.
  • ND composition required to achieve a therapeutically or prophylactically effective amount will vary from subject to subject, depending on species, age, and general condition of a subject, severity of the side effects, identity of the particular compound(s), mode of administration, and the like.
  • amount to be administered to, for example, a child or an adolescent can be determined by a medical practitioner or person skilled in the art and can be lower or the same as that administered to an adult.
  • a nanodegrader composition comprising a nanobody of any preceding aspect and a pharmaceutically acceptable carrier selected from an excipient, a diluent, a salt, a buffer, a stabilizer, a lipid, an emulsion, a nanoparticle, and a cream.
  • a pharmaceutically acceptable carrier selected from an excipient, a diluent, a salt, a buffer, a stabilizer, a lipid, an emulsion, a nanoparticle, and a cream.
  • One or more active agents e.g. the nanobody, the CPP, and the targeting peptide
  • Salts, esters, amides, prodrugs, and other derivatives of the active agents can be prepared using standards procedures known to those skilled in the art of synthetic organic chemistry and described, for example, by March (1992) Advanced Organic Chemistry; Reactions, Mechanisms, and Structure, 4 th Ed. N.Y. Wiley -Interscience.
  • the ND composition can be prepared as a “concentrate”, e.g. in a storage container of a premeasure volume and/or a predetermined amount ready for dilution, or in a soluble capsule ready for addition to a specified volume of water, saline, alcohol, hydrogen peroxide, or other diluent.
  • the ND composition is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more times.
  • the ND composition is administered daily.
  • the ND composition is administered weekly.
  • the ND composition is administered monthly.
  • the ND composition is administered yearly.
  • a method of degrading or eliminating a disease-related protein in a subject or biological sample comprising introducing a nanodegrader (ND) composition to the subject or biological sample, wherein the ND comprises a chimeric nanobody (Nb) and pharmaceutically acceptable carrier, wherein the Nb is operably linked to a cell penetrating peptide (CPP), a targeting peptide, and a proximity-enabled crosslinker (PEC), internalizing the ND into a cell of the subject or biological sample, wherein the CPP directs internalization into the cell, irreversibly targeting and binding the ND to the disease-related protein, wherein the PEC binds the disease-related protein to the ND, and degrading the disease- related protein, wherein the targeting peptide activates at least one proteolytic pathway to decrease the disease-related protein relative to a control subject or biological sample.
  • ND nanodegrader
  • a method of degrading or eliminating a disease-related protein in a subject or biological sample comprising introducing a nanodegrader (ND) composition of any preceding aspect to the subject or biological sample, internalizing the ND into a cell of the subject or biological sample, wherein the CPP directs internalization into the cell, irreversibly targeting and binding the ND to the disease-related protein, wherein the PEC binds the disease-related protein to the ND, and degrading the disease-related protein, wherein the targeting peptide activates at least one proteolytic pathway to decrease the disease-related protein relative to a control subject or biological sample.
  • ND nanodegrader
  • Proteolysis refers to the cellular process of shedding/degrading proteins into smaller units such as, for example polypeptides, amino acids, carbon molecules, nitrogen molecules, and/or hydrogen molecules.
  • Proteolysis in mammalian cells occurs by several mechanisms including a ubiquitin-proteosome pathway and/or a lysosomal proteolytic pathway.
  • Ubiquitin-proteosomal proteolysis occurs by the following steps: (1) covalent attachment of at least one ubiquitin molecule to a targeted protein, such as for example a misfolded protein and/or a dysfunctional protein; and (2) degradation of the targeted protein by the 26S proteosome complex with the release of free and reusable ubiquitin.
  • E3 ligase is the enzyme responsible for attaching the ubiquitin to the targeted protein(s).
  • the Nb is conjugated with E3 ligase ligands to catalyze the attachment of ubiquitin to targeted proteins.
  • E3 ligase ligands for PROTACs (Ishida et al. “E3 Ligase Ligands for PROTACS: How They Were Found and How to Discover New Ones” SLAS Discov. April 2021; 26(4): 484-502).
  • Proteolysis also occurs by lysosomal degradation wherein targeted proteins, such as for example misfolded proteins and/or dysfunctional proteins, are trafficked into cellular vesicles, called lysosomes, for degradation.
  • Lysosomes are acidic, membrane-bound cytoplasmic organelles found within various types of cells that can degrade various biomolecules, including proteins. Trafficking of targeted proteins into lysosomes occurs by macroautophagy, chaperone- mediated autophagy, endocytosis, or pinocytosis.
  • Jackson et al. is incorporated by reference herein for its teachings of degrading misfolded proteins by lysosomes (Jackson et al. “Cellular proteostasis: Degradation of misfolded proteins by lysosomes” Essays Biochem. October 15, 2016; 60(2): 173-180).
  • the method of any preceding aspect comprises a Nb comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, or a variant thereof
  • the method of any preceding aspect comprises a ND comprising a CPP with SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, or a variant thereof
  • the at least one proteolytic pathway comprises a ubiquitin- proteosome pathway, a lysosomal proteolytic pathway, or a combination thereof.
  • the method treats or prevents a disease of any preceding aspect.
  • the biological sample comprises a tissue biopsy, a cerebrospinal fluid (CSF) sample, a blood sample, a serum sample, or an isolated cell.
  • the biological sample is in vitro or in vivo.
  • the isolated cell is a neuron, a cancer cell, or an immune cell (including, but not limited to a B cell, T cell, and natural killer (NK) cell).
  • the present disclosure also provides methods to identify potential therapeutic compositions for diseases, including but not limited to Parkinson’s disease, comprising human neuronal co-culturing a therapeutic composition with neurons with a-synuclein (a-syn) A35T mutations or neurons treated with recombinant a-syn short fibrils, and identifying therapeutic compositions.
  • diseases including but not limited to Parkinson’s disease
  • a-syn a-synuclein
  • the present invention seeks to advance the therapeutic landscape for cancers by targeting but not limited to the selective degradation of the aggressive forms of the oncogenes: EGFR, HER2, CMTM6, cMYC, PCNA, HIF-la.
  • TPD proteolysis targeting chimeras
  • PROTACs proteolysis targeting chimeras
  • UPS ubiquitin-proteasome system
  • POI protein of interest
  • nanobody the smallest-known functional antibody fragment ( ⁇ 15 kDa) that is able to be delivered into living cells, have demonstrated its significant translational functionality in both preclinical and clinical studies.
  • Several intracellular nanobody-based TPD approaches have been developed but they require a fusion of nanobody with an E3 ligase catalytic domain or a subunit of the E3 ligase complex that causes unexpected cell toxicity.
  • a method exploiting the efficiency of nanobody and PROTAC is developed to approach close together POI and proteasome machinery to stimulate rapid POI degradation: nano-degraders.
  • the present disclosure provides the development of an intein-mediated expressed protein ligation (EPL) to site- selectively conjugate a nanobody (Nb) with small E3 ligase selective ligands.
  • EPL expressed protein ligation
  • Nb nanobody
  • small E3 ligase selective ligands small E3 ligase selective ligands.
  • the ALFA tag-specific nanobody was used for developing EPL approach.
  • a 25-mer peptide (Nb aa 98-122) was ligated to the intein-mediated Nb (aal-97) thioester fragment to produce full length ALFA Nb that was shown >95% ligation yield (Figure 7C). It has been contemplated that the E3 ligase complex can attach ubiquitin to the Nb Lysine residues and drives its proteasomal degradation quickly.
  • non-Lysine ALFA Nb variant was generated and shown functional as the wild type ( Figure 7D). It was also planned to introduce proteaseresistant D-amino acids into Nb via the EPL strategy to enhance its half-life.
  • the established EPL is used to generate nano-degraders specific to misfolded TDP-43 or Tau.
  • a crucial step for the success of this method is the delivery of the nano-degraders into the cells.
  • the nano-degraders are decorated with a highly efficient cyclic D-Arginine-rich CPP12 (Figure 7B) that was proved to deliver Nbs into live cells and mice (See, Zhang, W., et al., An intracellular nanobody targeting T4SS effector inhibits Ehrlichia infection.
  • MTD membrane translocation domain
  • Example 2 Development of nanobody based protein degraders targeting misfolded proteins in neurodegenerative disease.
  • Neurodegenerative diseases including Alzheimer's and Parkinson’s diseases, amyotrophic lateral sclerosis (ALS), prion diseases, and among others affect millions of people in the US.
  • ALS amyotrophic lateral sclerosis
  • prion diseases and among others affect millions of people in the US.
  • One of the earliest steps in the triggers of these diseases is the misfolding, aggregation, and accumulation of proteins in the central nervous system (CNS).
  • CNS central nervous system
  • a novel protein degrader is developed targeting misfolded proteins in human CNS cell models for neurodegenerative disorders.
  • ALS and tauopathies with two of the most common spectrum of TAR-DNA binding protein 43 (TDP-43) and Tau pathologies in neurodegenerative diseases, are characterized by altered protein clearance and deposits.
  • TDP-43 TAR-DNA binding protein 43
  • Tau pathologies in neurodegenerative diseases are characterized by altered protein clearance and deposits.
  • the present disclosure uses nano-degraders to target TDP-43 and Tau aggregates.
  • the human CNS cells differentiated from induced pluripotent stem cells (iPSCs) models is used to validate the nano-degrader method by analyzing its effects on the aggregate clearance and alleviate cellular pathological feature of human CNS cells.
  • This invention yields promising chemical biology strategies to counteract protein aggregates-induced cellular toxicity not only in ALS and Tauopathies but other misfolded protein disorders.
  • the present disclosure also provides human iPSCs systems with either ALS -associated TDP-43 Q33 IK mutant, ALS/FTD-linked Ubiquilin2 P497H/P506T mutant or Parkinson diseaselink a-synuclein A53T mutant differentiated into CNS cell types including upper or lower motor neurons, microglia, and astrocytes using our established protocol (Figure 1 and Figure 8A).
  • the expression, biochemical properties, cellular localization of protein aggregates are analyzed in iPSC-derived cells ( Figure 9 and Figure 10).
  • the effects on cellular clearance of aggregated forms induced by nano-degraders are characterized in these human cells. It is also tested whether this method ameliorates the cellular pathological features including the abnormalities in morphology and function of human CNS cell with these disease-associated mutations (Figure 11).
  • nano-degrader allows for the extraction of important information from biological systems, especially for the undruggable proteome and specific conformation of targets.
  • this technology is emerging as a highly versatile tool since nanobodies against every POI can be easily modified to become nanodegraders.
  • iPSCs models is significantly extended to the studies of the realm of human CNS cells.
  • Protein pathology is a hallmark of neurodegenerative diseases including PD, ALS and prion diseases and causes neuronal dysfunction and death.
  • Tauopathies, a-synucleinopathies, and TDP43 proteinopathies are some of the most common neurodegenerative disorders. Mutations in disease-associated genes such as TDP43 (TARDBPQ331K, Figure 2A) and Ubiquilin2 (UBQLN2P497H/P506T, Figure 2B), and a-syn (SNCAA53T, Figure 2C) have been identified to cause familial forms of ALS, ALS/FTD and PD, respectively. The mutations cause pathological aggregates and disrupt the ability of cellular machinery to clear the misfolded proteins.
  • CJD Creutzfeldt-Jakob disease
  • PrPC Cellular prion protein
  • PBNPE200K inherited CJD PBNPE200K, Figure 2D
  • PrPSc The neurotoxicity caused by prions (PrPSc), the misfolded form of PrPC, is related to the activation of specific signal transduction pathways.
  • Immunoassays of mature neurons indicated increased protein levels and aggregation of disease-hallmark proteins, including TDP43, a-syn, UBQLN2, prion protein, and some pathological phosphor-forms in mutant lines ( Figure 2).
  • Mouse models and patient tissues showed increased levels of phospho tau proteins which co-localized to misfolded protein and deposited in the protein inclusions.
  • Visualizing misfolded proteins by immunofluorescence (IF) display accumulates and co-localizes with paired helix filament (PHF) Tau in mutant neurons within the soma and dendritic regions ( Figures 2A, Cl and D2).
  • Synapse loss in neurodegeneration is caused by protein aggregate formation. Synaptic abnormalities can be observed before diagnosis in certain patients with the diseases, showing that protracted preclinical structure/function alterations play a key role in disease pathogenesis. Some membrane channel subunits may be retained intracellularly, as a result of their misfolded forms being affected by the mutations. This may cause abnormalities in synaptic protein composition, trafficking and localization, and lead to alterations in synaptic morphology and/or connectivity.
  • the model system allows for access to the neurons at the level of synapses. Visualizing spine morphology, a significant reduction of dendritic spine number was observed in mutant neurons (Figure 3). These data show that mutation may exert an effect on synaptic elements.
  • Nanobody the smallest-known functional antibody fragment, has demonstrated its significant translational potential in both preclinical and clinical studies.
  • chemical biology tools ( Figure 1A) were developed for targeting misfolded proteins, named nano-crosslinkers (NCs) and nano-degraders (NDs), to study protein interactomes and networks, and therapeutically eliminate them in human disease neuronal conditions.
  • the expressed protein ligation (EPL) method was established and these tools were designed utilizing three major technologies. First, Nbs are used to replace conventional antibodies to facilitate cell penetration. Second, the engineered Nb chimeras irreversibly react with the protein of interest (POI) by covalent interaction to overcome relatively low binding affinity and minimize off-target effects.
  • POI protein of interest
  • a cellpenetrating peptide CPP
  • CMA chaperon-mediated autophagy
  • MC macrocyclic
  • E3 ligands are conjugated to the Nbs to promote the internalization and targeted degradation.
  • This strategy has been successfully tested with the artificial ALFA tag Nb (Nb ALFA ) by creating the NC ALFA ( Figures 4A and 4B) and ND ALFA ( Figures 4C, 4D, 4E, and 4F) that target ALFA tagged-EFGP endogenously expressed in Hela cells and human neurons.
  • Nb-based Proximity Crosslinkers By adding a proximity-enabled covalent bonding of Nbs with POIs to improve their on-target retention, thereby enhancing the spatial resolution level of other chemical species such as photocrosslinker diazirine incorporated into the Nbs.
  • Proximity enabled sulfur-fluoride exchange (SuFEx) covalent linking is a suitable chemistry since the fluorosulfate warhead is biocompatible and able to react with multiple natural residues under physiological conditions (Figure 4A).
  • the EPL strategy was used to incorporate fluorosulfate-containing amino acids (FS-AAs) into Nb ALFA , which revealed a proximity-enabled X-linking with ALFA-tagged proteins.
  • NC ALFA is successfully internalized into neurons, then targeted and cross-linked with expressed ALFA-tagged EGFP in live neurons.
  • the X-linking product of NC ALFA and ALFA-tagged EGFP was successfully isolated by co-IP ( Figure 4B) in live neurons, this result proves the promising advantage of this method in studying protein complexes and PPIs.
  • the click chemistry strategy was used to incorporate FS-AAs into the Nb N-terminus (Fig 12).
  • Nb-based Degraders ALFA tag nanodegrader (ND ALFA ) was successfully developed ( Figures 4C, 4D, 4E, and 4F) that induced a complete degradation of ALFA-EGFP in Hela ( Figures 5A, 5B, and 5C) and human neurons ( Figures 5D, 5E, and 5F).
  • the specific targeting of ND ALFA was observed at the dendritic spine locations of neurons ( Figure 5D, right panels) which is an advantage for targeting supersaturated/misfolded synapse proteins.
  • the tunable Nb-based systems developed herein enable targeted monitoring of how protein regulation drives cellular signaling.
  • proteasome-targeting motifs such as peptide sequences (i.e. targeting VHL, Mdm2 E3 ligases) or E3 ligase-specific small molecules (Cereblon, VHL, IAPS, Mdm2, etc. ).
  • EPL Eligation-activated ligase
  • PrPC The physiological cellular form PrPC is present on the outside leaflet of the membrane of most cell types. In neurons, PrPC is predominant in axons and dendrites. PrPC involves several cellular processes, including neuritogenesis, neuronal homeostasis, cell signaling, cell adhesion, and a protective role against stress. PrPC serves as cellular receptors for b-sheet-rich neurotoxic proteins including a-syn and TDP43.
  • UBQLN2 protein structure contains four stress-induced protein 1 (STI-l)-like domains, which are involved in the interaction with heat shock proteins and autophagy mediators. Mutation in these domains also causes ALS.
  • STI-l stress-induced protein 1
  • STI1 protein is found as a cell surface ligand for PrPC and their interaction triggers neuroprotection.
  • the staining of spinal cord (Sc) sections contained large irregularshaped structures in the gray matter that immunoreactivity with PrP antibodies. PrP staining is positive to Ubiquitin.
  • UBQLN2 inclusions which are positive to PrP, are also found colocalized with aggregated TDP43. Data shows that PrP constitutes ubiquitinated proteins with amyloid conformations. Then, the effect on PrPC degradation under the expression of UBQLN2P497H was analyzed in the N2a cell line, an overexpressed PrP cell line. A delay in the degradation rate of PrPC affected by the P497H mutation was observed. Co-immunoprecipitation assay of PrP and UBQLN2 proteins in Hela shows intermolecular interaction between PrP and mutant UBQLN2 proteins. More important, cells and neurons with UBQLN2 mutation increase the appearance of cytoplasmic PrP.
  • the conventional proximity biotin-labeling methods do not provide direct information about PPIs.
  • Other antibody -based mapping techniques such as pMap have only been applied for cell membrane surfaces since it is challenging to deliver such large antibodies into the cells.
  • more controllable photo-crosslinking reactions also have been used to capture the physical interactome.
  • the incorporation of photo-X-linkers into target proteins using amber codon suppression remains a challenge due to cytotoxicity from proteome- wide nonspecific incorporation.
  • genetically encoded tags such as Halo, Snap and others only recruit a single photo-X-linker to target protein, therefore limiting their capturing ability.
  • NDs targeting TDP43 aggregates have been developed using the scFV domains derived from heavy chain (VH) and light chain (VL) of the anti-TDP43 aggregate antiboby 3B12A.
  • VH heavy chain
  • VL light chain
  • multi-milligrams of anti-TDP-43 VH domain in fusion with lysosome-targeting CMA1 tag have been purified from 1 L of E. coli expression.
  • an N-terminal fusion with the VH domain and the E. coli periplasmic signal peptide PelB was proved to be helpful in enhancing the solubility of the VL domain.
  • a similar protein chemical approach described above has been used to make NDS TDP43 from purified VH and VL domains.
  • Example 3 Targeted degradation of oncogenic EGFR and HER2 using nanobodies as proximity-directing agents.
  • bioPROTACs biological PROTACs
  • AbsTAC antibody-based PROTAC
  • LYTACs lysosomal-targeting chimeras
  • LYTACs LYTACs
  • the presence of the Fc region in LYTACs and the intricacy of attaching glycans can impact immune responses and pose challenges in manufacturing and scalability.
  • Many existing bioPROTACs work via intracellular overexpression, limiting them to the cytoplasm, and can hinder target accessibility, increase cytotoxicity, reduce engineering control, and prevent modulation of the tumor environment or impact multiple cells.
  • nano-degraders targeting some key oncogenic membrane proteins such as EGFR and HER2.
  • CKLF like MARVEL transmembrane domain-containing 6 (CMTM6) overexpression that has been proposed to confer a resistance to the FDA-approved immunotherapy using trastuzumab antibody in HER2- positive breast cancers.
  • SKBR3, MDA-MB-231, MCF7, and HEPG2 cell lines will be used to evaluate the delivery efficiency and degradation potency of NDs.
  • Luciferase complementation assay, NanoLuc will be employed to determine the delivery efficiency and endosomal escape of the NDs. It will be evaluated by fusing HiBit, an 11 -ami no acid peptide from NanoLuc. This involves transfecting the cells with plasmid DNA encoding LgBiT, an 18 kDa subunit of NanoLuc. Successful cytosolic delivery and escape results in HiBit binding to LgBiT, forming an active luciferase, NanoLuc. The degradation activity would be analyzed following NDs incubation for 24 hours in varying doses.
  • immunoblot will contribute to assessing the impact of ND treatment on the levels of downstream effectors, specifically phosphorylated form of pAkt and pERKl/2.
  • Quantitative mass spectrometry by Tandem Mass Tag will be employed to confirm the specificity of degradation across the proteome under NDs treatment.
  • Specific 26S proteasome inhibitors such as MG132 or Bortezomib, would be used to assess if clearance by NDs is via the proteasome.
  • assays following manufacturer protocols to evaluate their antiproliferative, migration effect (Incucyte S3 Live-Cell Analysis System) and cell viability by luminometer (CellTiter-Glo Luminescent Cell Viability reagent).
  • transwell migration assays to assess cell migration.
  • Cells will be seeded on a porous membrane dividing two chambers, using 10% FBS in the lower chamber as a chemoattractant. After incubation, non-migrated cells will be cleared, and migrated cells on the membrane's bottom side will be fixed with 4% PF A, stained, and quantified to assess migration.
  • PEG polyethylene glycol
  • n refers to number of PEG or G.
  • SEQ ID NO: 33 SEQ ID NO: 33 - chaperone-mediated autophagy 2 (CMA2) peptide motif KFERQKILDQRFFE
  • SEQ ID NO: 34 Cell-penetrating peptide (CPP)
  • SEQ ID NO: 35 Cell-penetrating peptide (CPP) rRrGrKkRr wherein r refers to D-Arginine and k refers to D-lysine.
  • CPP Cell-penetrating peptide
  • SEQ ID NO: 36 Cell-penetrating peptide (CPP)
  • RrRrRrRrRrRr wherein r refers to D-Arginine.

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Abstract

The present disclosure relates compositions comprising and/or encoding nanodegraders (ND) and methods of use thereof.

Description

NANOBODY-BASED PROTEIN DEGRADERS AND RELATED METHODS
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority to, and the benefit of, U.S. Provisional Patent Application No. 63/490,151, filed March 14, 2023, entitled “NANOBODY-BASED PROTEIN DEGRADERS AND RELATED METHODS,” and U.S. Provisional Patent Application No. 63/593,669, filed October 27, 2023, entitled “NANOBODY-BASED PROTEIN DEGRADERS AND RELATED METHODS,” which are incorporated by reference herein in their entireties.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
This invention was made with Government Support under Grant No. CA241105 awarded by the National Institutes of Health. The Government has certain rights in the invention.
REFERENCE TO SEQUENCE LISTING
The sequence listing submitted on March 14th, 2024, as an .XML file entitled “103361- 452WO1_ST26” created on March 12, 2024, and having a file size of 50,901 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5).
FIELD
The present disclosure relates compositions comprising and/or encoding nanodegraders and methods of use thereof.
BACKGROUND
Protein folding diseases, including Alzheimer's and Parkinson’s diseases, amyotrophic lateral sclerosis (ALS), prion diseases, and among others affect millions of people in the US. Unfortunately, there is currently no adequate treatment for these diseases. One of the earliest steps in the triggers of these diseases is the misfolding, aggregation, and accumulation of proteins in the central nervous system (CNS). Thus, what is needed in the art is selective elimination of misfolded proteins as a therapeutic strategy to mitigate protein folding diseases, including but not limited to neurodegenerative diseases, inflammatory diseases, infectious diseases, and cancer. The compounds, compositions, and methods disclosed herein address these and other needs. SUMMARY
The present disclosure provides compositions, vectors, and kits comprising a chimeric nanobody protein degrader. The present disclosure provides methods of using compositions, vectors, and kits comprising a chimeric nanobody protein degrader. The present disclosure also provides methods of treating or preventing a disease in a subject in need thereof. The present disclosure also provides methods of decreasing or reducing a disease-related protein.
In one aspect, disclosed herein is a nanodegrader (ND) composition comprising a chimeric nanobody (Nb) and a pharmaceutically acceptable carrier, wherein the Nb is operably linked to a cell penetrating peptide (CPP), a targeting peptide, and a proximity-enabled crosslinker (PEC).
In some embodiments, the Nb comprises less than 200 amino acids or is less than 15kDa. In some embodiments, the Nb does not comprise a lysine residue. In some embodiments, the Nb comprises at least one protease-resistant D-amino acid. In some embodiments, the Nb comprises the Nb comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, or a variant thereof.
In some embodiments, the CPP comprises a cyclic CPP or a membrane translocation domain (MTD). In some embodiments, the CPP comprises an arginine-rich cyclic CPP. In some embodiments, the CPP comprises CPP12, or a variant thereof. In some embodiments, the CPP comprises SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, or a variant thereof.
In some embodiments, the targeting peptide comprises a chaperon-mediated autophagy (CMA) peptide, a macrocyclic (MC) peptide, E3 ligase ligand, or derivatives thereof. In some embodiments, the PEC comprises a proximity-enabled sulfur-fluoride exchange (SuFEx) crosslinker.
In some embodiments, the Nb is irreversibly bound to a protein of interest (POI) by the PEC. In some embodiments, the POI includes, but is not limited to a misfolded protein or a cancer- associated protein selected from tau, TAR DNA-binding protein 43 (TDP-43), a-synucleic, P- amyloid, prion proteins, human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR), CKLF like MARVEL transmembrane domain-containing 6 (CMTM6), human transcription factor cellular MYC (c-MYC), proliferating cell nuclear antigen (PCNA), and hypoxia-inducible factor 1-alpha (HIF-la).
In some embodiments, the pharmaceutically acceptable carrier comprises an excipient, a diluent, a salt, a buffer, a stabilizer, a lipid, an emulsion, or a native Nb. In some embodiments, the ND further comprises one or more protein labeling compounds. In some embodiments, the protein labeling compound comprises biotin, a reporter enzyme, a fluorophore, or a radioactive isotope.
In one aspect, disclosed herein is a vector comprising a nucleic acid sequence encoding the ND, or a fragment thereof, of any preceding aspect.
In one aspect, disclosed herein is a kit comprising the ND, the vector, or a salt thereof, of any preceding aspect. In some embodiments, the kit further comprises one or more reagents for stabilizing the ND. In some embodiments, the kit further comprises one or more reagents for delivering the ND to a biological sample or subject. In some embodiments, the kit further comprises one or more reagents for amplifying the vector. In some embodiments, the kit further comprises one or more reagents for expressing the vector in a cell.
In one aspect, disclosed herein is a method of treating or preventing a disease in a subject in need thereof, the method comprises administering a pharmaceutically effective amount of a nanodegrader (ND) composition comprising a chimeric nanobody (Nb) of any preceding aspect and a pharmaceutically acceptable carrier, wherein the Nb is operably linked to a cell penetrating peptide (CPP), a targeting peptide, a proximity-enabled crosslinker (PEC), and wherein the ND targets and degrades a disease-related protein.
In one aspect, disclosed herein is a method of treating or preventing a disease in a subject in need thereof, the method comprises administering a pharmaceutically effective amount of the nanodegrader (ND) composition of any preceding aspect.
In some embodiments, the method decreases or reduces the disease-related protein relative to an untreated control. In some embodiments, the disease-related protein comprises tau, TAR DNA-binding protein 43 (TDP-43), a-synucleic, P-amyloid, a prion protein, human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR), CKLF like MARVEL transmembrane domain-containing 6 (CMTM6), human transcription factor cellular MYC (c- MYC), proliferating cell nuclear antigen (PCNA), or hypoxia-inducible factor 1-alpha (HIF-la).
In some embodiments, the method further comprises administering the ND and a therapeutic agent. In some embodiments, the therapeutic agent comprises an antibiotic, an antiinflammatory compound, a sedative, an anesthetic, an anti-viral agent, a peptide hormone, an antidiabetic agent, a steroid, or combinations thereof.
In some embodiments, the disease comprises a neurodegenerative disease, a cancer, including but not limited to Alzheimer’s disease, ataxia, Huntington’s disease, Parkinson’s disease, amyotrophic lateral sclerosis (ALS), Friedreich ataxia, Lewy body disease, a prion disease, Creutzfeldt-Jakob disease, neurological cancers (including, but not limited to brain cancers), lung cancers, breast cancers, and gastric cancers.
In one aspect, disclosed herein is a method of degrading or eliminating a disease-related protein in a subject or biological sample, the method comprising introducing a nanodegrader (ND) composition to the subject or biological sample, wherein the ND comprises a chimeric nanobody (Nb) of any preceding aspect and pharmaceutically acceptable carrier, wherein the Nb is operably linked to a cell penetrating peptide (CPP), a targeting peptide, and a proximity-enabled crosslinker (PEC), internalizing the ND into a cell of the subject or biological sample, wherein the CPP directs internalization into the cell, irreversibly targeting and binding the ND to the disease-related protein, wherein the PEC binds the disease-related protein to the ND, and degrading the disease- related protein, wherein the targeting peptide activates at least one proteolytic pathway to decrease the disease-related protein relative to a control subject or biological sample.
In one aspect, disclosed herein is a method of degrading or eliminating a disease-related protein in a subject or biological sample, the method comprising introducing a nanodegrader (ND) composition of any preceding aspect to the subject or biological sample, internalizing the ND into a cell of the subject or biological sample, wherein the CPP directs internalization into the cell, irreversibly targeting and binding the ND to the disease-related protein, wherein the PEC binds the disease-related protein to the ND, and degrading the disease-related protein, wherein the targeting peptide activates at least one proteolytic pathway to decrease the disease-related protein relative to a control subject or biological sample.
In some embodiments, the method of any preceding aspect comprises a Nb comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, or a variant thereof. In some embodiments, the method of any preceding aspect comprises a ND comprising a CPP with SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, or a variant thereof.
In some embodiments, the PEC comprises a proximity-enabled sulfur-fluoride exchange (SuFEx) crosslinker. In some embodiments, the Nb is irreversibly bound to the disease-related protein of any preceding aspect by the PEC. In some embodiments, the pharmaceutically acceptable carrier comprises an excipient, a diluent, a salt, a buffer, a stabilizer, a lipid, an emulsion, or a native Nb. In some embodiments, the method of any preceding aspect comprises one or more protein labeling compounds of any preceding aspect. In some embodiments, the at least one proteolytic pathway comprises a ubiquitin- proteosome pathway, a lysosomal proteolytic pathway, or a combination thereof.
In some embodiments, the method treats or prevents a disease selected from Alzheimer’s disease, ataxia, Huntington’s disease, Parkinson’s disease, amyotrophic lateral sclerosis (ALS), Friedreich ataxia, Lewy body disease, a prion disease, or Creutzfeldt-Jakob disease.
In some embodiments, the biological sample comprises a tissue biopsy, a cerebrospinal fluid (CSF) sample, a blood sample, a serum sample, or an isolated cell.
BRIEF DESCRIPTION OF FIGURES
The accompanying figures, which are incorporated in and constitute a part of this specification, illustrate several aspects described below.
Figures 1A, IB, and 1C show a platform technology. Figure 1A shows that targeting misfolded proteins or cancer-associated proteins are developed and applied on specialized human neuronal models of diseases of Figure IB. Figure 1A shows a diagram of nanobodybased protein molecules, including nano-degrader (ND) and nano-crosslinker (NC). Nanobody (Nb) is attached with CPP (cell-penetrating peptide), targeting sequences (such as CMA, chaperon mediated autophagy, MC peptides) and chemical species (such as TMR, Tetramethyl- rhodamine fluorophore), biotin and proximity-enabled covalent crosslinkers. Figure IB shows a two-layer human iPSC-derived neuronal culture system. Functioning human neurons (Figure B2), with a representative result of single- neuron patch-clamp recordings (Figure Bl), are cultured on coverslips suspended over a feeder layer of glial cells (Figure B3, microglia). Immunofluorescence imaging of mature neurons and microglia staining for cell type markers including Tuj 1 (Class III B-Tubulin), Vglutl (Vesicular glutamate transporter 1), and Ibal (Ionized calcium-binding adaptor molecule 1). Figure 1C shows the Nb on the ND/NCs recognizes and covalently crosslinks with targets (disease-associated proteins) at the membrane and/or in the cytoplasm via cross- linker fluorosulfonate amino acids (FS-AAs). CPP peptide promotes the internalization of ND/NCs into neurons. The ND-targeted protein complexes are led by CMA peptides and/or E3 ligands/MC peptides to the lysosomal and/or proteasome systems for degradation.
Figures 2A, 2B, 2C, and 2D show the protein pathology in human disease neuronal models with mutation, (Figure 2A) ALS- TARDBPQ331K, (Figure 2B) ALS/FTD-UBQLN2P497H, (Figure 2C) PD-SNCAA53T and (Figure 2D) CJD-PRNPE200K. Immunoassay shows the disease hallmark accumulation of pathogenic proteins in human neuronal cultures with mutations, including misallocated TDP43 (Figures 2A and 2B), ubiquilin2 (Figure 2B), a-syn (Figure 2C), prion protein (Figure 2D), and pathological Tau (PHF Tau). Plots show the quantifications from pool measurements of 2-4 replicated cultures, 2-3 sub-clones. White arrows indicated the protein inclusions. The used antibodies are shown. Mutant neurons with TARDBPQ331K, SCNAA53T and UBQLN P497H/P506T are derived from iisogenic IPSCs. The neurons with PRNPF200K are derived from patient IPSCs. PK: proteinase K treatment. ScN2a, a prion-infected cell line derived from N2a. SMI-31 : neuro-filament maker. D2 shows neurofibrillary tau tangles-like pathology in PRNPE200K neurons.
Figures 3A and 3B show the mutant neurons display a lower number of dendritic spines compared to normal neurons. Figures 3A shows that the cultures were stained with fluorescent phalloidin (red) for F-actin. Spines were visualized and quantified. Figure 3B shows that the pooled measurements of staining were collected from 30-40 regions from 2-3 replicated cultures.
Figures 4A, 4B, 4C, 4D, 4E, and 4F show the development of NCs. Figure 4A shows a schematic for the proximity-enabled cross-linking of Nb with POI-protein of interest using SuFex chemistry with fluorosulfate amino acids FS-Tyrosine (FSY) and FS-Lysine (K-FS) incorporated into Nb. Figure 4B shows that NCALFA was treated on ALFA-eGFP- expressed live neurons. Immunoprecipitation (IP) assay with Dynabeads and anti-Flag antibody. Blot with anti HA-tag antibody to detect both NCALFA and ALFA-eGFP proteins shows the successful cross-linking of NCALFA and eGFP-ALFA protein in the lysate (input) and IP samples. FT: flow though. Development of NDs (Figures 4C, 4D, 4E, and 4F). Figure 4C shows a schematic for ND with the attachments of CPP, CMA and chemical species, including TMR fluorophore. Figure 4D shows the SDS gel for the conjugation of semisynthetic CMA-NbALFA with TMR. Figure 4E shows the SDS gel for conjugating CMA- NbALFA-TMR with CPP peptide. Figure 4F shows the duel function NDs strategies. Schematic of site- selective conjugation of Nb containing N-ter Cysteine (Cys) with Proteasome-directing agents (Pro) N-hydroxysuccinimide (NHS)-ester. Conjugation SDS gel of NDs with attachment of Pro, including modified MC 1/2 peptides (lane 1&2) or the von Hippel-Lindau peptide motif (VHL), an E3 ligand (lane 3&4).
Figures 5 A, 5B, 5C, 5D, 5E, 5F, 5G, 5H, and 51 show the successful degradation of ALFA- eGFP by NDALFA in Hela cells (Figures 5A, 5B, and 5C) and neurons (Figures 5D, 5E, and 5F). IF images (Figure 5A) of eGFP expressed-Hela pretreated with CHX and exposed to NDALFA (red). Blots (Figure 5B) show the degradation of eGFP in Hela. eGFP and NDALFA were detected by anti-HA and -biotin antibodies, respectively. IF images of eGFP-expressed neurons exposed to NbALFA (Figure 5D and E). NDALFA was decorated with TMR (red), and biotin. Arrows indicated co-localization of ND and eGFP in the cells. Quantifications show a significant decrease in eGFP level in ND-treated cell (Figure 5C) and neuron (Figure 5F) samples. Data is from pool measurements of 3 different cultures. NDs target a-syn (Figures 5G, 5H, and 51), NDa-syn2. C]\jA-NbSyn2-CPP-TMR. (G) SDS gel for the conjugation of semisynthetic CMA-Nb“' syn, lane 1 and 2 (NbSyn2), lane 3 and 4 (NbSyn87), with CPP peptide via a disulfide bond. Figure 5H shows the cellular internalization and a-syn degradation of NDs“'syn2 (red, TMR) in Hela cells expressed a-syn proteins (green) (aggregate A53T a-syn protein indicated by arrows). Arrow heads show co-localization of ND“'syn2 molecules and a-syn proteins. Figure 51 shows the quantifications of a-syn intensity shows reducing of signal of ND treated samples compare to non-treated samples.
Figures 6A, 6B, 6C, and 6D show the studying of PrP interactomes. Figure 6A shows the SDS gel for the purification of PrP nanobody (NbPrP) and conjugation of NbPrP with CPP. Figure 6C and 6D shows the interaction of NbPrP-based proteins (shown in panel F i gure 6 A), including NbPrP (Figure 6C) and NbPrP-CPP (Figure 6D), with PrP in Hela. Hela cells overexpressing PrP (Figure 6C) or both PrP and UBQLN2P497H (Figure 6D) were treat with NbPrP or NbPrP-CPP for 2 hours. IF staining show co-localization NbPrP- based proteins (green) with membrane PrP (Cl, red) and cytoplasmic PrP (DI, red). NCPrP strategy with live ineurons at membrane (C2) and cytoplasm (D2). The proximity-enabled cross-linking of NCPrP with PrPc using SuFex chemistry with fluorosulfate amino acids incorporated into NbPrP. Protein mapping with UV light and diazirine-NbPrP are used to generate reactive carbenes that cross-link nearby proteins within 1 nm radius (green area). Selecting labeling occurs through immunoreaction of photocatalyst-NCPrP. Figure 6B shows the photo-proximity protein work flow on live neurons.
Figures 7A, 7B, 7C, and 7D show the development of nano-degrader. Figure 7A shows the bifunctional nano-degrader brings together El, E2, E3 ligase complex and POI resulting in proteasomal degradation of POI. Figure 7B shows the schematic for the generation of nanodegrader contains E3 ligase ligand, POI-specific nanobody using EPL, followed by the attachment of cell-permeable peptide (CPP). Figure 7C shows the SDS-PAGE analysis for EPL of recombinant Nb(l-97)-Intein fusion without (lane 1, control) and with the synthetic Nb(98-122) peptide (lane 2). Figure 7D shows the pulldown assay for GST-ALFA _Nb fusion proteins: wild type (WT-Nb, left) and non-Lysine variant (KR-Nb, right) immobilized on glutathione beads and then mixed with ALFA-tagged sortase A (ALFA-SrtA). The beads were washed and then analyzed by SDS-gel.
Figures 8A and 8B show the differentiation of human iPSCs into neurons and microglial cells. An example of misfolded Tau protein in prion diseases, has been characterized in iPSC- derived cell models. Accumulation of pathological Tau (green), the paired helical filaments (PHF- Tau) aggregated forms (white arrows), in human IPSC-derived neurons from patients carrying E200K mutation of prion protein, which causes Creutzfeldt-Jakob Disease (a familial prion disease in human), as compared to the non-carrier (NC).
Figures 9A, 9B and 9C show the level of total a-synuclein (a-syn) protein after the treatment with ND21 on human neuron contain A53T mutation. Figure 9A shows the schematic for the generation of nanodegrader 21 (ND21) contains CMA1 peptide, syn 2 nanobody using EPL with malamide (Ma) conjugation, followed by the attachment of cell-permeable peptide (CPP) CR10. Figures 9B and 9C show IPSCs- derived human neuron with A53T a-syn mutation at day 28 of differentiation, which were used to test the ND21. Figure 9B shows the Western Blot results of neurons were treated with ND21 at 0.5uM final concentration for 32 and 96 hours (A53T + ND21). Neurons in the same batch were treated with same amount of PBS buffer as a control (A53T). Wild-type neurons at the same day-old were used as a control. After the treatments, neuron cultures were collected for Western blotting. The proteins were detected by Western blot analysis using antibodies shown. Figure 9C show graphical data of total asyn protein level in the lysate samples in this experiment. Data shown is the mean ± SEM.
Figures 10A, 10B and IOC show the internalization and a-syn degradation effects of the ND21 in the soma region of human neurons. IPSCs- derived human neuron with A53T asyn mutation at day 28 of differentiation were used to test the ND21. Figure 10B shows neurons were treated with ND21 at 0.5uM final concentration for 32 hours (A53T + ND21). Figure 10A shows neurons in the same batch were treated with same amount of PBS buffer as control (A53T). Figures 10 A and B show neuron cultures after these treatments, which were fixed and stained for a-syn (green) and HA tag (red). ND21 treated a-syn A53T neurons show reduction of a-syn protein level compared to the non-treated samples. ND21, which is positive to HA staining in treated neurons, is located in the soma regions (Bl). The boxed regions in each panel are shown at higher magnification in the smaller horizontal panels to the right (Al A2 and Bl, B2). Figure 10C shows the quantitation of cytoplasmic a-syn integrated density levels show the fold change of a-syn in the soma regions of these neurons. Pooled measurements were collected from 15-20 neurons from 3 different cultures. Data shown is the mean ± SEM.
Figures 11A, 1 IB and 11C show the rescue effects of ND21 on synaptotoxicity of human neurons with a-syn A53T mutation. IPSCs- derived human neuron with A53T asyn mutation at day 28 of differentiation were used to test the ND21. Figure 1 IB shows neurons were treated with ND21 at 0.5uM final concentration for 32 hours (A53T + ND21). Figure 11A shows neurons in the same batch were treated with same amount of PBS buffer as control (A53T). Figures 11 A and 1 IB show neuron cultures after the treatments which were fixed and stained with Phalloidin (red) forF-Actinto visualize the dendritic spine morphologies. ND21 treated asyn A53T neurons shown increasing number of spines compare to the non-treated samples. The boxed regions (Al, Bl) in each panel are shown at higher magnification in the smaller horizontal panels to the right (Al, Bl). Figure 11C shows the quantitation of spine number shows number of dendritic spines per lOpM. Pooled measurements were collected from dendri sections of 15-20 neurons from 3 different cultures. Data shown is the mean ± SEM.
Figures 12A, 12B, 12C show the schematic illustration for using copper-catalyzed azidealkyne cycloaddition (Cu-AAC) to incorporate proximity-enabled crosslinker fluorosulfonate (FSY) amino acids into Nanobodies (Nbs). Of note, the FSY can be incorporated into the Lysine side chain directly or via a variable PEG(n>2) linker.
Figures 13A, 13B, 13C, and 13D show schematic illustrations for genetic code expansion. Figure 13 A shows the schematic illustration for genetic code expansion with the amber codon (TAG) suppression to incorporate 4-Azido Phenylalanine (Azido-Phe) into Nanobodies (Nbs). One plasmid expressing the tRNA and its cognate aminoacyl-tRNA-synthetase (aaRS) that has been evolved to incorporate Azido-Phe (magenta star). Another plasmid containing the gene of nanobody with the amber codon (TAG) that is recognized by the cognate charged tRNA. Once these plasmids have been introduced in the cells, the Azido-Phe can be incorporated using the existing protein translation machinery. Figure 13B shows the copper-free azdie-alkyne cycloaddition for Alfa-tag Nb containing Azido-Phe with dibenzocyclooctyne (DBCO) sulfo-Cy5 (fluorophore probe, Lumiprobe). Left panel is for coomassie staining and right panel is for Cy5 fluorescence scanning at 700 nm wavelength. M: protein marker. Figure 13C shows the peptide carrier (5) for use with click chemistry to incorporate E3 ligase ligands into Nbs. Figure 13D show the copper-free azide-DBCO cycloaddition for Azido-Phe-Nb with E3 -ligand-linked DBCO- peptide.
Figures 14A, 14B, and 14C show illustrations of ND delivery and degradation mechanism. Figure 14A illustrates the NDs cellular delivery and degradation mechanisms. Figure 14B shows the SDS-PAGE analysis of CMA1 developed HER2-ND conjugated with CPP. Figure 14C shows the immunofluorescent image confirming the colocalization of Nb-CMAl construct and HER2 in SKBR3 cells.
Figure 15 shows that vector map of the pET-22b-PelB-TEV-HA-Nanobody-3C-6xHis plasmid.
Figure 16 shows the vector map of the pET28-TEV-Cys-HA-Nanobody-3C-6xHis plasmid.
Figure 17 shows the vector map of the pTXBl-TEV-C-HA-Nanobody-CMAl plasmid. Figures 18 A, 18B, 18C, 18D, and 18E show the various nanodegrader constructs. Figure 18A shows the ectodomain-targeting nanobody. Figure 18B shows the covalent binding nanodegrader (ND). Figure 18C shows the cytoplasmic-targeting nanobody. Figure 18D shows the biparatopic ND. Figure 18E shows the proteasome-targeting ND.
Figures 19 shows the cell-penetrating peptides (CPPS) used herein. The CPPs of the present disclosure include Di-TAMRA-TAT peptide comprising SEQ ID NO: 34, or a variant thereof, Cys-cyclic-TAT peptide comprising SEQ ID NO: 35, or a variant thereof, or Cys-cyclic- Deca-Arginine peptide comprising SEQ ID NO: 36, or a variant thereof.
Figure 20 shows the peptide (SEQ ID NO: 2), or a variant thereof, used for incorporation of proximity-enabled crosslinker fluorosulfonate (FSY) into nanobodies using protein semi synthesis strategy.
DETAILED DESCRIPTION
The following description of the disclosure is provided as an enabling teaching of the disclosure in its best, currently known embodiment(s). To this end, those skilled in the relevant art will recognize and appreciate that many changes can be made to the various embodiments of the invention described herein, while still obtaining the beneficial results of the present disclosure. It will also be apparent that some of the desired benefits of the present disclosure can be obtained by selecting some of the features of the present disclosure without utilizing other features. Accordingly, those who work in the art will recognize that many modifications and adaptations to the present disclosure are possible and can even be desirable in certain circumstances and are a part of the present disclosure. Thus, the following description is provided as illustrative of the principles of the present disclosure and not in limitation thereof.
Reference will now be made in detail to the embodiments of the invention, examples of which are illustrated in the drawings and the examples. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.
Terminology
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. The term “comprising” and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various embodiments, the terms “consisting essentially of’ and “consisting of’ can be used in place of “comprising” and “including” to provide for more specific embodiments and are also disclosed. As used in this disclosure and in the appended claims, the singular forms “a”, “an”, “the”, include plural referents unless the context clearly dictates otherwise.
The following definitions are provided for the full understanding of terms used in this specification.
Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that when a value is disclosed that “less than or equal to” the value, “greater than or equal to the value” and possible ranges between values are also disclosed, as appropriately understood by the skilled artisan. For example, if the value “10” is disclosed the “less than or equal to 10” as well as “greater than or equal to 10” is also disclosed. It is also understood that the throughout the application, data is provided in a number of different formats, and that this data, represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point “10” and a particular data point 15 are disclosed, it is understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15 are considered disclosed as well as between 10 and 15. It is also understood that each unit between two particular units are also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.
The terms "about" and "approximately" are defined as being “close to” as understood by one of ordinary skill in the art. In one non-limiting embodiment the terms are defined to be within 10%. In another non-limiting embodiment, the terms are defined to be within 5%. In still another non-limiting embodiment, the terms are defined to be within 1%.
As used herein, the terms "may," "optionally," and "may optionally" are used interchangeably and are meant to include cases in which the condition occurs as well as cases in which the condition does not occur. Thus, for example, the statement that a formulation "may include an excipient" is meant to include cases in which the formulation includes an excipient as well as cases in which the formulation does not include an excipient. “Composition” refers to any agent that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition. The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, a vector, polynucleotide, cells, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like. When the term “composition” is used, then, or when a particular composition is specifically identified, it is to be understood that the term includes the composition per se as well as pharmaceutically acceptable, pharmacologically active vector, polynucleotide, salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc.
"Comprising" is intended to mean that the compositions, methods, etc. include the recited elements, but do not exclude others. "Consisting essentially of' when used to define compositions and methods, shall mean including the recited elements, but excluding other elements of any essential significance to the combination. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives, and the like. "Consisting of' shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions provided and/or claimed in this disclosure. Embodiments defined by each of these transition terms are within the scope of this disclosure.
An "increase" can refer to any change that results in a greater amount of a symptom, disease, composition, condition, or activity. An increase can be any individual, median, or average increase in a condition, symptom, activity, composition in a statistically significant amount. Thus, the increase can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% increase so long as the increase is statistically significant.
A "decrease" can refer to any change that results in a smaller amount of a symptom, disease, composition, condition, or activity. A substance is also understood to decrease the genetic output of a gene when the genetic output of the gene product with the substance is less relative to the output of the gene product without the substance. Also, for example, a decrease can be a change in the symptoms of a disorder such that the symptoms are less than previously observed. A decrease can be any individual, median, or average decrease in a condition, symptom, activity, composition in a statistically significant amount. Thus, the decrease can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% decrease so long as the decrease is statistically significant.
By “reduce” or other forms of the word, such as “reducing” or “reduction,” means lowering of an event or characteristic (e.g., misfolded protein aggregation). It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to. For example, “reduces protein misfolding” means reducing the amount of misfolded protein relative to a standard or a control.
By “prevent” or other forms of the word, such as “preventing” or “prevention,” is meant to stop a particular event or characteristic, to stabilize or delay the development or progression of a particular event or characteristic, or to minimize the chances that a particular event or characteristic will occur. Prevent does not require comparison to a control as it is typically more absolute than, for example, reduce. As used herein, something could be reduced but not prevented, but something that is reduced could also be prevented. Likewise, something could be prevented but not reduced, but something that is prevented could also be reduced. It is understood that where reduce or prevent are used, unless specifically indicated otherwise, the use of the other word is also expressly disclosed.
The terms “treat,” “treating,” and grammatical variations thereof as used herein, include partially or completely delaying, alleviating, mitigating or reducing the intensity of one or more attendant symptoms of a disorder or condition and/or alleviating, mitigating or impeding one or more causes of a disorder or condition. Treatments according to the disclosure may be applied preventively, prophylactically, palliatively or remedially. Treatments are administered to a subject prior to onset (e.g., before obvious signs of neurodegeneration), during early onset (e.g., upon initial signs and symptoms of neurodegeneration), or after an established development of neurodegenerati on .
The term “subject” refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. In one aspect, the subject can be human, non-human primate, bovine, equine, porcine, canine, or feline. The subject can also be a guinea pig, rat, hamster, rabbit, mouse, or mole. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician.
The term “amino acid,” includes but is not limited to amino acids contained in the group consisting of alanine (Ala or A), cysteine (Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), phenylalanine (Phe or F), glycine (Gly or G), histidine (His or H), isoleucine (He or I), lysine (Lys or K), leucine (Leu or L), methionine (Met or M), asparagine (Asn or N), proline (Pro or P), glutamine (Gin or Q), arginine (Arg or R), serine (Ser or S), threonine (Thr or T), valine (Vai or V), tryptophan (Trp or W), and tyrosine (Tyr or Y) residues. The term “amino acid residue” also may include amino acid residues contained in the group consisting of homocysteine, 2- Aminoadipic acid, N-Ethylasparagine, 3 -Aminoadipic acid, Hydroxylysine, P-alanine, P- Aminopropionic acid, allo-Hydroxylysine acid, 2- Aminobutyric acid, 3-Hydroxyproline, 4- Aminobutyric acid, 4-Hydroxyproline, piperidinic acid, 6-Aminocaproic acid, Isodesmosine, 2- Aminoheptanoic acid, allo-Isoleucine, 2-Aminoisobutyric acid, N-Methylglycine, sarcosine, 3- Aminoisobutyric acid, N-Methylisoleucine, 2-Aminopimelic acid, 6-N-Methyllysine, 2,4- Diaminobutyric acid, N-Methylvaline, Desmosine, Norvaline, 2,2'-Diaminopimelic acid, Norleucine, 2,3-Diaminopropionic acid, Ornithine, and N-Ethylglycine. Typically, the amide linkages of the peptides are formed from an amino group of the backbone of one amino acid and a carboxyl group of the backbone of another amino acid.
Reference also is made herein to peptides, polypeptides, proteins, and compositions comprising peptides, polypeptides, and proteins. As used herein, a polypeptide and/or protein is defined as a polymer of amino acids, typically of length>100 amino acids (Garrett & Grisham, Biochemistry, 2nd edition, 1999, Brooks/Cole, 110). A peptide is defined as a short polymer of amino acids, of a length typically of 20 or less amino acids, and more typically of a length of 12 or less amino acids (Garrett & Grisham, Biochemistry, 2nd edition, 1999, Brooks/Cole, 110).
The peptides, polypeptides, and proteins disclosed herein may be modified to include nonamino acid moieties. Modifications may include but are not limited to carboxylation (e.g., N- terminal carboxylation via addition of a di-carboxylic acid having 4-7 straight-chain or branched carbon atoms, such as glutaric acid, succinic acid, adipic acid, and 4,4-dimethylglutaric acid), amidation (e.g., C-terminal amidation via addition of an amide or substituted amide such as alkylamide or dialkylamide), PEGylation (e.g., N-terminal or C-terminal PEGylation via additional of polyethylene glycol), acylation (e.g., O-acylation (esters), N-acylation (amides), S- acylation (thioesters)), acetylation (e.g., the addition of an acetyl group, either at the N-terminus of the protein or at lysine residues), formylation lipoylation (e.g., attachment of a lipoate, a C8 functional group), myristoylation (e.g., attachment of myristate, a C14 saturated acid), palmitoylation (e.g., attachment of palmitate, a C16 saturated acid), alkylation (e.g., the addition of an alkyl group, such as an methyl at a lysine or arginine residue), isoprenylation or prenylation (e.g., the addition of an isoprenoid group such as farnesol or geranylgeraniol), amidation at C- terminus, glycosylation (e.g., the addition of a glycosyl group to either asparagine, hydroxylysine, serine, or threonine, resulting in a glycoprotein). Distinct from glycation, which is regarded as a nonenzymatic attachment of sugars, polysialylation (e.g., the addition of poly sialic acid), glypiation (e.g., glycosylphosphatidylinositol (GPI) anchor formation, hydroxylation, iodination (e.g., of thyroid hormones), and phosphorylation (e.g., the addition of a phosphate group, usually to serine, tyrosine, threonine, or histidine).
It should also be noted that amino acids, and derivatives (with exception of glycine) occurs in two isomeric forms: L-forms or D-forms. The L- and D- forms represent the same atoms of an amino acid, however the atoms can have different arrangements, which can impact the amino acid properties and functions. The two forms are similar in that they both occur naturally and comprise a central carbon atom, at least one hydrogen atom, a carboxylic group, an amine group, and a variable group. The two forms differ in that they are usually mirrored images of each other, wherein the location of the amine group varies. L-amino acids are used in protein synthesis, while D-amino acid are less common in protein synthesis. L-amino acids rotate counterclockwise or to the left in a process known as levorotation. D-amino acids rotate clockwise or to the right in a process known as dextrorotation.
A “fusion protein” refers to a protein formed by the fusion of at least one peptide, polypeptide, protein or variant thereof as disclosed herein to at least one molecule of a heterologous peptide, polypeptide, protein or variant thereof. The heterologous protein(s) may be fused at the N-terminus, the C-terminus, or both termini. A fusion protein comprises at least a fragment or variant of the heterologous protein(s) that are fused with one another, preferably by genetic fusion (i.e., the fusion protein is generated by translation of a nucleic acid in which a polynucleotide encoding all or a portion of a first heterologous protein is joined in-frame with a polynucleotide encoding all or a portion of a second heterologous protein). The heterologous protein(s), once part of the fusion protein, may each be referred to herein as a “portion”, “region” or “moiety” of the fusion protein.
The term “variant” means a polypeptide derived from a parent polypeptide by one or more (several) alteration(s), i.e., a substitution, insertion, and/or deletion, at one or more (several) positions. A substitution means a replacement of an amino acid occupying a position with a different amino acid; a deletion means removal of an amino acid occupying a position; and an insertion means adding 1 or more, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably 1-3 amino acids immediately adjacent an amino acid occupying a position. In relation to substitutions, ‘immediately adjacent’ may be to the N-side (‘'upstream’) or C-side (‘downstream’ ) of the amino acid occupying a position (‘the named amino acid'). Therefore, for an amino acid named/numbered ‘X,’ the insertion may be at position ‘ X+T (‘downstream’) or at position ‘X--1 ’ (‘upstream').
A “variant” of a particular polypeptide sequence may be defined as a polypeptide sequence having at least 50% sequence identity to the particular polypeptide sequence over a certain length of one of the polypeptide sequences using blastp with the “BLAST 2 Sequences” tool available at the National Center for Biotechnology Information's website. (See Tatiana A. Tatusova, Thomas L. Madden (1999), “Blast 2 sequences — a new tool for comparing protein and nucleotide sequences”, FEMS Microbiol Lett. 174:247-250). In some embodiments a variant polypeptide may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to a reference polypeptide.
A variant polypeptide may have substantially the same functional activity as a reference polypeptide. For example, a variant polypeptide may exhibit or more biological activities associated with binding a ligand and/or binding DNA at a specific binding site.
A “nucleotide” is a compound consisting of a nucleoside, which consists of a nitrogenous base and a 5-carbon sugar, linked to a phosphate group forming the basic structural unit of nucleic acids, such as DNA or RNA. The four types of nucleotides are adenine (A), cytosine (C), guanine (G), and thymine (T), each of which are bound together by a phosphodiester bond to form a nucleic acid molecule.
A “nucleic acid” is a chemical compound that serves as the primary information-carrying molecules in cells and make up the cellular genetic material. Nucleic acids comprise nucleotides, which are the monomers made of a 5-carbon sugar (usually ribose or deoxyribose), a phosphate group, and a nitrogenous base. A nucleic acid can also be a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA). A chimeric nucleic acid comprises two or more of the same kind of nucleic acid fused together to form one compound comprising genetic material.
A “full length” polynucleotide sequence is one containing at least a translation initiation codon (e.g., methionine) followed by an open reading frame and a translation termination codon. A “full length” polynucleotide sequence encodes a “full length” polypeptide sequence.
A “variant,” “mutant,” or “derivative” of a particular nucleic acid sequence may be defined as a nucleic acid sequence having at least 50% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the “BLAST 2 Sequences” tool available at the National Center for Biotechnology Information's website. (See Tatiana A. Tatusova, Thomas L. Madden (1999), “Blast 2 sequences — a new tool for comparing protein and nucleotide sequences”, FEMS Microbiol Lett. 174:247-250). In some embodiments a variant polynucleotide may show, for example, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length relative to a reference polynucleotide.
The word “vector” refers to any vehicle that carries a polynucleotide into a cell for the expression of the polynucleotide in the cell. The vector may be, for example, a plasmid, a virus, a phage particle, or a nanoparticle. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may in some instances, integrate into the genome itself. In some embodiments, the vector is a DNA construct containing a DNA sequence which is operably linked to a suitable control sequence capable of effecting the expression of the DNA in a suitable host cell. Such control sequences can include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites, and sequences which control the termination of transcription and translation. A plasmid or a viral vector can be capable of extrachromosomal replication or, optionally, can integrate into the host genome. As used herein, the term "integrated" used in reference to an expression vector (e.g., a plasmid or viral vector) means the expression vector, or a portion thereof, is incorporated (physically inserted or ligated) into the chromosomal DNA of a host cell. As used herein, a “viral vector” refers to a virus-like particle containing genetic material which can be introduced into a eukaryotic cell without causing substantial pathogenic effects to the eukaryotic cell. A wide range of viruses or viral vectors can be used for transduction but should be compatible with the cell type the virus or viral vector are transduced into (e.g., low toxicity, capability to enter cells). Suitable viruses and viral vectors include adenovirus, lentivirus, retrovirus, among others. In some embodiments, the expression vector encoding a chimeric polypeptide is a naked DNA or is comprised in a nanoparticle (e.g., liposomal vesicle, porous silicon nanoparticle, gold-DNA conjugate particle, polyethyleneimine polymer particle, cationic peptides, etc.). In other embodiments, the vector is a lipid nanoparticle. Lipid nanoparticles can be used to deliver mRNA to a host cell for expression of the mRNA in the host cell.
The term “administer,” “administering”, or derivatives thereof refer to delivering a composition, substance, inhibitor, or medication to a subject or object by one or more the following routes: oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-joint, parenteral, intra-arteriole, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation or via an implanted reservoir. The term “parenteral” includes subcutaneous, intravenous, intramuscular, intraarticular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional, and intracranial injections or infusion techniques. The term “kit” describes a wide variety of bags, containers, carrying cases, and other portable enclosures which may be used to carry and store solid substances, liquid substances, and other accessories necessary to stabilize, administering, store, and/or express the compositions and vectors disclosed herein.
"Pharmaceutically acceptable carrier" (sometimes referred to as a “carrier”) means a carrier or excipient that is useful in preparing a pharmaceutical or therapeutic composition that is generally safe and non-toxic, and includes a carrier that is acceptable for veterinary and/or human pharmaceutical or therapeutic use. The terms "carrier" or "pharmaceutically acceptable carrier" can include, but are not limited to, phosphate buffered saline solution, water, emulsions (such as an oil/water or water/oil emulsion) and/or various types of wetting agents.
As used herein, the term “carrier” encompasses any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations. The choice of a carrier for use in a composition will depend upon the intended route of administration for the composition. The preparation of pharmaceutically acceptable carriers and formulations containing these materials is described in, e.g., Remington's Pharmaceutical Sciences, 21st Edition, ed. University of the Sciences in Philadelphia, Lippincott, Williams & Wilkins, Philadelphia, PA, 2005. Examples of physiologically acceptable carriers include saline, glycerol, DMSO, buffers such as phosphate buffers, citrate buffer, and buffers with other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN (ICI, Inc.; Bridgewater, New Jersey), polyethylene glycol (PEG), and PLURONICS (BASF; Florham Park, NJ). To provide for the administration of such dosages for the desired therapeutic treatment, compositions disclosed herein can advantageously comprise between about 0.1% and 99% by weight of the total of one or more of the subject compounds based on the weight of the total composition including carrier or diluent.
Compositions and Compounds
Abnormal increases and dysfunctional post-translational processing of proteins (often leading to misfolded proteins) lead to various diseases including, but not limited to neurogenerative diseases and disorders, cancer, and autoimmune diseases. Neurodegenerative diseases currently have no cure despite many efforts to advance traditional pharmacology approaches. The diseases caused by the presence and accumulation of pathological proteins lead to progressive loss of structure and/or function of neurons, including the death of neurons. Clearance of misfolded proteins is a biological process tightly coupled with neurotoxicity. One strategy for preventing misfolded proteins is targeted protein degradation (TPD), which include development of nanobody-based protein degraders (ND). TPD strategies are desirable to overcome inherent off-target effects from genetic strategies CRISPR/Cas9 and RNA interference. For instance, the PROTAC method employs the ubiquitin-proteasome system (UPS), a major intracellular protein degradation system, to degrade a protein of interest (POI). However, this method has been only applied to targets with available small molecule inhibitors, and classically “undruggable” proteins remain challenging. Particularly, this strategy also proves unworkable for targeting misfolded proteins in neurodegenerative diseases. Herein therapeutic compositions comprising NDs are developed and tested herein for targeting pathogenic proteins toward intracellular degradation processes.
The present disclosure provides compositions, vectors, and kits comprising a chimeric nanobody protein degrader. The present disclosure provides methods of using compositions, vectors, and kits comprising a chimeric nanobody protein degrader.
The present disclosure provides therapeutic molecules targeting the degradation of pathological proteins. The Targeted Protein Degradation (TPD) system is called nanobody-based protein degraders (nano-degraders, NDs). Relying on the proximity enabled cross-linking reaction, The inventors create chemically modified nanobody chimeras that irreversibly crosslink with the protein while the conjugated cell-penetrating peptide (CPP) and chaperon-mediated autophagy (CMA) peptides and/or small E3 ligase selective ligands allow rapid degradation of targeted protein in lysosomes and/or proteasome. The resultant NDs will be employed to target pathological proteins in protein misfolding diseases and cancers.
The present disclosure also provides disease-associated neuronal culture models derived from human IPSCs, which successfully recapitulate the diseases with several key pathological features, including the accumulation of pathological proteins, to assess the rescue efficiency of the NDs in a realm of human context.
Disclosed herein is a covalently engineered nanobody chimeras that irreversibly react with the protein while the conjugated cell-penetrating peptide (CPP) and chaperon-mediated autophagy (CMA) peptides allow rapid targeted protein degradation in lysosomes. ND molecules are synthesized and characterized that target cellular pathological a-syn proteins and 2) it is demonstrated that ND degrades pathological a-syn and moderates the neurotoxic effects in the human PD-a-syn neuronal cultures.
In one aspect, disclosed herein is a nanodegrader (ND) composition comprising a chimeric nanobody (Nb) and a pharmaceutically acceptable carrier, wherein the Nb is operably linked to a cell penetrating peptide (CPP), a targeting peptide, and a proximity-enabled crosslinker (PEC).
Nanobodies (Nb) are currently the smallest antibody molecules with a molecular weight of 1/10 of a normal antibody. In addition to the antigenic reactivity of monoclonal antibodies, nano-antibodies possess unique functional properties, such as small molecular mass, strong stability, good solubility, easy expression, weak immunogenicity, strong penetrability, strong targeting, simple in humanization, low in preparation cost, etc. Nb use often improves the shortcomings of traditional antibody long-term development cycle, low stability, and harsh storage conditions.
As used herein, the term “nanobody” or “immunoglobulin” is a protein of approximately 15,000 Dalton (15kDa, unconjugated) with the same structural features of antibodies, consisting of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to the heavy chain through a covalent disulfide bond, and the number of disulfide bonds between the heavy chains of different immunoglobulin isoforms is different. Each heavy and light chain also has intra-chain disulfide bonds which are regularly spaced. Each heavy chain has a variable region (VH) at one end followed by a plurality of constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other end; the constant region of the light chain is opposite to the first constant region of the heavy chain, and the variable region of the light chain is opposite to the variable region of the heavy chain. Special amino acid residues form an interface between the variable regions of the light and heavy chains.
The disclosure also provides other proteins or fusion expression products of the ND of the invention. Specifically, the present disclosure includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a heavy chain containing a variable region, if the variable region is identical or at least 90% identical, preferably at least 95% identical to the heavy chain of the nanobody of the present disclosure.
In general, the antigen-binding properties of a nanobody can be described by three specific regions located in the variable region of the heavy chain, referred as variable regions (CDRs), and the segment is divided into four frame regions (FRs). The amino acid sequences of four FRs are conservative and do not directly participate in binding reactions. These CDRs form a loop structure in which the P-sheets formed by the FRs therebetween are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen- binding site of the nanobody. The amino acid sequences of the same type of nanobodies can be compared to determine which amino acids constitute the FR or CDR regions.
The variable regions of the heavy chains of the nanobodies of the invention become a particular interest because at least a part of them is involved in binding antigens. Thus, the present invention includes those molecules having a nanobody heavy chain variable region with a CDR, provided that their CDRs are 90% or more (preferably 95% or more, the most preferably 98% or more) identical to the CDRs identified herein.
The present disclosure includes not only intact nanobodies but also fragment(s) of immunologically active nanobody or fusion protein(s) formed from nanobodies with other sequences. Therefore, the present disclosure also includes fragments, derivatives, and analogs of the nanobodies.
As used herein, the terms “fragment,” “derivative,” and “analog” refer to a polypeptide that substantially retains the same biological function or activity of a nanobody of the invention. Polypeptide fragments, derivatives or analogs of the invention may be (i) polypeptides having one or more conservative or non-conservative amino acid residues (preferably non-conservative amino acid residues) substituted. Such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) a polypeptide having a substituent group in one or more amino acid residues; or (iii) a polypeptide formed by fusing a mature polypeptide and another compound (such as a compound that increases the half-life of the polypeptide, for example, polyethylene glycol); or (iv) a polypeptide formed by fusing an additional amino acid sequence to the polypeptide sequence (e.g., a leader or secretory sequence or a sequence used to purify this polypeptide or a proprotein sequence, or a fusion protein formed with a 6 His tag). According to the teachings herein, these fragments, derivatives, and analogs are within the scope of one of ordinary skill in the art.
The nanobody of the present disclosure refers to a polypeptide including the CDR regions. The term also encompasses variant forms of polypeptides comprising the above CDR regions that have the same function as the nanobodies of the invention. These variations include, but are not limited to, deletion insertions and/or substitutions of one or several (usually 1-50, preferably 1-30, more preferably 1-20, optimally 1-10) amino acids, and addition of one or several (generally less than 20, preferably less than 10, and more preferably less than 5) amino acids at C-terminus and/or N-terminus. For example, in the art, the substitution of amino acids with analogic or similar properties usually does not alter the function of the protein. For another example, addition of one or several amino acids at the C-terminus and/or N-terminus usually does not change the function of the protein. The term also includes active fragments and active derivatives of the nanobodies of the invention.
The variant forms of the polypeptide include homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNAs capable of hybridizing with DNA encoding the nanobody of the present invention under high or low stringent conditions, and polypeptides or proteins obtained using antiserum against the nanobodies of the invention.
The invention also provides other polypeptides, such as a fusion protein comprising nanobodies or fragments thereof. In addition to almost full-length polypeptides, the present invention also includes fragments of the nanobodies of the invention. Typically, the fragment has at least about 50 contiguous amino acids of the nanobody of the invention, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids.
In the present disclosure, “a conservative variant of a nanobody of the present invention” refers to the polypeptides in which there are up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids substituted by amino acids having analogical or similar properties, compared to the amino acid sequence of the nanobody of the present invention.
The nanobodies herein may be used alone or in combination or conjugated with a detectable marker (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modification moiety, or a combination thereof.
In some embodiments, the Nb comprises less than 200 amino acids or is less than 15kDa. In some embodiments, the Nb comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,
46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,
72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136,
137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155,
156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174,
175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193,
194, 195, 196, 197, 198, 199, 200 amino acids. In some embodiments, the Nb is 0.5kDa, 1 kDa, 2 kDa, 3kDa, 4kDa, 5kDa, 6kDa, 7kDa, 8kDa, 9kDa, lOkDa, l lkDa, 12kDa, 13kDa, 14kDa, 15kDa.
Natural L-amino acids serve as the starting components for protein synthesis and protein- related research. D-amino acids share identical chemical and physical properties with L-amino acids, however D-amino acids rotate plane polarized light in the opposite direction relative to L- amino acids. In regards to protein-related research, peptides are increasingly becoming attractive drug candidate to treat various diseases. However, it has become recognized that peptides containing L-amino acids a highly susceptible to protein degradation. It has become recognized that peptides containing D-amino acids are more resistant to endogenous protein degradation. Thus, the present disclosure provides an ND composition comprising peptides with at least one D-amino acid, wherein the D-amino acids prevent protein degradation of the components of the ND. In some embodiments, the Nb comprises at least one protease-resistant D-amino acid.
In some embodiments, the Nb comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, or a variant thereof.
In some embodiments, the CPP comprises a cyclic CPP or a membrane translocation domain (MTD). In some embodiments, the CPP comprises an arginine-rich cyclic CPP. In some embodiments, the CPP comprises CPP12, or a variant thereof. In some embodiments, the CPP comprises SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, or a variant thereof.
In some embodiments, the CPP comprises the constructs including but not limited to Di- TAMRA-TAT peptide comprising SEQ ID NO: 34, or a variant thereof; Cys-cyclic-TAT peptide comprising SEQ ID NO: 35, or a variant thereof; or Cys-cyclic-Deca-Arginine peptide comprising SEQ ID NO: 36, or a variant thereof. In some embodiments, the CPP comprises any one of the CPP of Figure 19, or a variant thereof.
The ubiquitin-proteosome pathway is a selective protein degradation pathway that uses ubiquitin (a 76 amino acid polypeptide to mark proteins for degradation. Proteins are marked for degradation by the attachment of ubiquitin to lysine residues. To prevent degradation of ND compositions, the present disclosure provides ND compositions having minimal or completely eliminating lysine residues. In some embodiments, the Nb does not comprise a lysine residue.
The NDs herein may optionally have ubiquitin-recruiting ligase (E3 ligase, E3 ubiquitin ligase) binding moiety (binder). The ubiquitin ligase binder may be an analog of thalidomide, which binds the E3 ubiquinase known as cereblon. The ubiquitin ligase binder may also be a ligand that binds the von Hippel-Lindau tumor suppressor (VHL) protein, which is attached via a linker to another small molecule (target- moiety) that binds a target protein (see, e.g., Lai et cd.. Angew. Chem. Int. Ed. Engl. 55(22): 807-810 (2016). Targeted protein degradation refers to small molecule (e.g. , protein binding small molecule) induced ubiquitination and degradation of disease targets, in which the small molecule may simultaneously recruit both a ubiquitin ligase and the target protein into proximity of each other, which may lead to ubiquitination of the target protein.
Protein degradation is a highly regulated and essential process that maintains cellular homeostasis. The selective identification and removal of damaged, misfolded, or excess proteins is achieved via the ubiquitin-proteasome pathway (UPP). The UPP in fact is central to the regulation of almost all cellular processes, including antigen processing, apoptosis, biogenesis of organelles, cell cycling, DNA transcription and repair, differentiation and development, immune response and inflammation, neural and muscular degeneration, morphogenesis of neural networks, modulation of cell surface receptors, ion channels and the secretory pathway, the response to stress and extracellular modulators, ribosome biogenesis and viral infection.
Covalent attachment of multiple ubiquitin molecules by an E3 ubiquitin ligase to a terminal lysine residue marks the protein for proteasome degradation, where the protein is digested into small peptides and eventually into its constituent amino acids that serve as building blocks for new proteins. Defective proteasomal degradation has been linked to a variety of clinical disorders including Alzheimer's disease, Parkinson's disease, Huntington's disease, muscular dystrophies, cardiovascular disease, and cancer among others.
There are over 600 E3 ubiquitin ligases which facilitate the ubiquitination of different proteins in vivo, which can be divided into four families: HECT-domain E3s, U-box E3s, monomeric RING E3s and multi-subunit E3s. See generally Li et al. (PLOS One, 2008, 3, 1487) titled “Genome-wide and functional annotation of human E3 ubiquitin ligases identifies MULAN, a mitochondrial E3 that regulates the organelle's dynamics and signaling ”; Berndsen et al. (Nat. Struct. Mol. Biol., 2014, 21, 301-307) titled “New insights into ubiquitin E3 ligase mechanism”; Deshaies et al. (Ann. Rev. Biochem., 2009, 78, 399-434) titled “RING domain E3 ubiquitin ligases.”; Spratt et al. (Biochem. 2014, 458, 421-437) titled “RBR E3 ubiquitin ligases: new structures, new insights, new questions .”; and Wang et al. (Nat. Rev. Cancer., 2014, 14, 233-347) titled “Roles of F-box proteins in cancer.”
In some embodiments, the targeting peptide comprises a chaperon-mediated autophagy (CMA) peptide, a macrocyclic (MC) peptide, E3 ligase ligand, or derivatives thereof. In some embodiments, the targeting peptide comprises SEQ ID NO: 9, or a variant thereof. In some embodiments, the targeting peptide comprises SEQ ID NO: 33, or a variant thereof. In some embodiments, the targeting peptide comprises an amino acid sequence of a chaperon-mediated autophagy (CMA) peptide, a macrocyclic (MC) peptide, E3 ligase ligand, or derivatives thereof. In some embodiments, the PEC comprises a proximity-enabled sulfur-fluoride exchange (SuFEx) crosslinker. In some embodiments, the Nb is irreversibly bound to a protein of interest (POI) by the PEC. In some embodiments, the POI comprises a misfolded protein selected from tau, TAR DNA- binding protein 43 (TDP-43), a-synuclein, P-amyloid, or prion proteins. In some embodiments, the POI comprises an accumulated oncoprotein or a cancer-associated protein including, but not limited to human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR), CKLF like MARVEL transmembrane domain-containing 6 (CMTM6), human transcription factor cellular MYC (c-MYC), proliferating cell nuclear antigen (PCNA), and hypoxia-inducible factor 1 -alpha (HIF-la), immunosuppressive protein, cytokines, or chemokines including, but not limited to transforming growth factor-beta (TGF-P) and interleukin- 10 (IL-10), or variants thereof.
In some embodiments, the pharmaceutically acceptable carrier comprises an excipient, a diluent, a salt, a buffer, a stabilizer, a lipid, an emulsion, or a native Nb.
In some embodiments, the ND further comprises one or more protein labeling compounds. In some embodiments, the protein labeling compound comprises biotin, a reporter enzyme, a fluorophore, or a radioactive isotope.
In some embodiments, the fluorophore includes, but are not limited to, 1,5 IAEDANS; 1,8- ANS; 4- Methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein; 5-Carboxyfluorescein (5- FAM); 5-Carboxynapthofluorescein; 5-Carboxytetramethylrhodamine (5-TAMRA); 5-Hydroxy Tryptamine (5-HAT); 5-ROX (carboxy-X-rhodamine); 6-Carboxyrhodamine 6G; 6-CR 6G; 6- JOE; 7-Amino-4-methylcoumarin; 7- Aminoactinomycin D (7-AAD); 7-Hydroxy-4- I methylcoumarin; 9-Amino-6-chloro-2-methoxyacridine (ACMA); ABQ; Acid Fuchsin; Acridine Orange; Acridine Red; Acridine Yellow; Acriflavin; Acriflavin Feulgen SITSA; Aequorin (Photoprotein); AFPs - AutoFluorescent Protein - (Quantum Biotechnologies) see sgGFP, sgBFP; Alexa Fluor 350™; Alexa Fluor 430™; Alexa Fluor 488™; Alexa Fluor 532™; Alexa Fluor 546™; Alexa Fluor 568™; Alexa Fluor 594™; Alexa Fluor 633™; Alexa Fluor 647™; Alexa Fluor 660™; Alexa Fluor 680™; Alizarin Complexon; Alizarin Red; Allophycocyanin (APC); AMC, AMCA- S; Aminomethylcoumarin (AMCA); AMCA-X; Aminoactinomycin D; Aminocoumarin; Anilin Blue; Anthrocyl stearate; APC-Cy7; APTRA-BTC; APTS; Astrazon Brilliant Red 4G; Astrazon Orange R; Astrazon Red 6B; Astrazon Yellow 7 GLL; Atabrine; ATTO- TAG™ CBQCA; ATTOTAG™ FQ; Auramine; Aurophosphine G; Aurophosphine; BAO 9 (Bisaminophenyloxadiazole); BCECF (high pH); BCECF (low pH); Berberine Sulphate; Beta Lactamase; BFP blue shifted GFP (Y66H); Blue Fluorescent Protein; BFP/GFP FRET; Bimane; Bisbenzemide; Bisbenzimide (Hoechst); bis- BTC; BlancophorFFG; Blancophor SV; BOBO™ -1; BOBO™-3; Bodipy492/515; Bodipy493/503; Bodipy500/510; Bodipy; 505/515; Bodipy 530/550; Bodipy 542/563; Bodipy 558/568; Bodipy 564/570; Bodipy 576/589; Bodipy 581/591; Bodipy 630/650-X; Bodipy 650/665-X; Bodipy 665/676; Bodipy Fl; Bodipy FL ATP; Bodipy Fl-Ceramide; Bodipy R6G SE; Bodipy TMR; Bodipy TMR-X conjugate; Bodipy TMR-X, SE; Bodipy TR; Bodipy TR ATP; Bodipy TR-X SE; BO-PRO™ -1; BO-PRO™ -3; Brilliant Sulphoflavin FF; BTC; BTC-5N; Calcein; Calcein Blue; Calcium Crimson - ; Calcium Green; Calcium Green- 1 Ca2+ Dye; Calcium Green-2 Ca2+; Calcium Green-5N Ca2+; Calcium Green-C18 Ca2+; Calcium Orange; Calcofluor White; Carboxy-X-rhodamine (5-ROX); Cascade Blue™; Cascade Yellow; Catecholamine; CCF2 (GeneBlazer); CFDA; CFP (Cyan Fluorescent Protein); CFP/YFP FRET; Chlorophyll; Chromomycin A; Chromomycin A; CL-NERF; CMFDA; Coelenterazine; Coelenterazine cp; Coelenterazine f; Coelenterazine fcp; Coelenterazine h; Coelenterazine hep; Coelenterazine ip; Coelenterazine n; Coelenterazine O; Coumarin Phalloidin; C-phycocyanine; CPM I Methylcoumarin; CTC; CTC Formazan; Cy2™; Cy3.1 8; Cy3.5™; Cy3™; Cy5.1 8; Cy5.5™; Cy5™; Cy7™; Cyan GFP; cyclic AMP Fluorosensor (FiCRhR); Dabcyl; Dansyl; Dansyl Amine; Dansyl Cadaverine; Dansyl Chloride; Dansyl DHPE; Dansyl fluoride; DAPI; Dapoxyl; Dapoxyl 2; Dapoxyl 3’DCFDA; DCFH (Diehl orodihydrofluorescein Diacetate); DDAO; DHR (Dihydorhodamine 123); Di-4-ANEPPS; Di-8-ANEPPS (non-ratio); DiA (4-Di 16-ASP); Diehl orodihydrofluorescein Diacetate (DCFH); DiD- Lipophilic Tracer; DiD (DilC 18(5)); DIDS; Dihydorhodamine 123 (DHR); Dil (DilC18(3)); I Dinitrophenol; DiO (DiOC18(3)); DiR; DiR (DilC18(7)); DM-NERF (high pH); DNP; Dopamine; DsRed; DTAF; DY-630-NHS; DY-635- NHS; EBFP; ECFP; EGFP; ELF 97; Eosin; Erythrosin; Erythrosin ITC; Ethidium Bromide; Ethidium homodimer-1 (EthD-1); Euchrysin; EukoLight; Europium (111) chloride; EYFP; Fast Blue; FDA; Feulgen (Pararosaniline); FIF (Formaldehyde Induced Fluorescence); FITC; Flazo Orange; Fluo-3; Fluo-4; Fluorescein (FITC); Fluorescein Diacetate; Fluoro-Emerald; Fluoro-Gold (Hydroxystilbamidine); Fluor-Ruby; FluorX; FM 1-43™; FM 4-46; Fura Red™ (high pH); Fura Red™/Fluo-3; Fura-2; Fura-2/BCECF; Genacryl Brilliant Red B; Genacryl Brilliant Yellow 10GF; Genacryl Pink 3G; Genacryl Yellow 5GF; GeneBlazer; (CCF2); GFP (S65T); GFP red shifted (rsGFP); GFP wild type’ non-UV excitation (wtGFP); GFP wild type, UV excitation (wtGFP); GFPuv; Gloxalic Acid; Granular blue; Haematoporphyrin; Hoechst 33258; Hoechst 33342; Hoechst 34580; HPTS; Hydroxycoumarin; Hydroxystilbamidine (FluoroGold); Hydroxytryptamine; Indo-1, high calcium; Indo-1 low calcium; Indodicarbocyanine (DiD); Indotricarbocyanine (DiR); Intrawhite Cf; JC-1; JO JO-1; JO-PRO- 1; LaserPro; Laurodan; LDS 751 (DNA); LDS 751 (RNA); Leucophor PAF; Leucophor SF; Leucophor WS; Lissamine Rhodamine; Lissamine Rhodamine B; Calcein/Ethidium homodimer; LOLO-1; LO-PRO-1; ; Lucifer Yellow; Lyso Tracker Blue; Lyso Tracker Blue-White; Lyso Tracker Green; Lyso Tracker Red; Lyso Tracker Yellow; LysoSensor Blue; LysoSensor Green; LysoSensor Yellow/Blue; Mag Green; Magdala Red (Phloxin B); Mag-Fura Red; Mag-Fura-2; Mag-Fura-5; Mag-lndo-1; Magnesium Green; Magnesium Orange; Malachite Green; Marina Blue; I Maxiion Brilliant Flavin 10 GFF; Maxiion Brilliant Flavin 8 GFF; Merocyanin; Methoxy coumarin; Mitotracker Green FM; Mitotracker Orange; Mitotracker Red; Mitramycin; Monobromobimane; Monobromobimane (mBBr-GSH); Monochlorobimane; MPS (Methyl Green Pyronine Stilbene); NBD; NBD Amine; Nile Red; Nitrobenzoxedidole; Noradrenaline; Nuclear Fast Red; i Nuclear Yellow; Nylosan Brilliant lavin E8G; Oregon Green™; Oregon Green™ 488; Oregon Green™ 500; Oregon Green™ 514; Pacific Blue; Pararosaniline (Feulgen); PBFI; PE-Cy5; PE-Cy7; PerCP; PerCP-Cy5.5; PE- TexasRed (Red 613); Phloxin B (Magdala Red); Phorwite AR; Phorwite BKL; Phorwite Rev; Phorwite RPA; Phosphine 3R; PhotoResist; Phycoerythrin B [PE]; Phycoerythrin R [PE]; PKH26 (Sigma); PKH67; PMIA; Pontochrome Blue Black; POPO-1; POPO-3; PO-PRO-1; PO- 1 PRO- 3; Primuline; Procion Yellow; Propidium lodid (Pl); PyMPO; Pyrene; Pyronine; Pyronine B; Pyrozal Brilliant Flavin 7GF; QSY 7; Quinacrine Mustard; Resorufin; RH 414; Rhod-2; Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5 GLD; Rhodamine 6G; Rhodamine B; Rhodamine B 200; Rhodamine B extra; Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine Phallicidine; Rhodamine: Phalloidine; Rhodamine Red; Rhodamine WT; Rose Bengal; R-phy cocyanine; R-phycoerythrin (PE); rsGFP; S65A; S65C; S65L; S65T; Sapphire GFP; SBFI; Serotonin; Sevron Brilliant Red 2B; Sevron Brilliant Red 4G; Sevron I Brilliant Red B; Sevron Orange; Sevron Yellow L; sgBFP™ (super glow BFP); sgGFP™ (super glow GFP); SITS (Primuline; Stilbene Isothiosulphonic Acid); SNAFL calcein; SNAFL-1; SNAFL-2; SNARF calcein; SNARF 1; Sodium Green; SpectrumAqua; SpectrumGreen; SpectrumOrange; Spectrum Red; SPQ (6-methoxy- N-(3 sulfopropyl) quinolinium); Stilbene; Sulphorhodamine B and C; Sulphorhodamine Extra; SYTO 11; SYTO 12; SYTO 13; SYTO 14; SYTO 15; SYTO 16; SYTO 17; SYTO 18; SYTO 20; SYTO 21; SYTO 22; SYTO 23; SYTO 24; SYTO 25; SYTO 40; SYTO 41; SYTO 42; SYTO 43; SYTO 44; SYTO 45; SYTO 59; SYTO 60; SYTO 61; SYTO 62; SYTO 63; SYTO 64; SYTO 80; SYTO 81; SYTO 82; SYTO 83; SYTO 84; SYTO 85; SYTOX Blue; SYTOX Green; SYTOX Orange; Tetracycline; Tetramethylrhodamine (TRITC); Texas Red™; Texas Red-X™ conjugate; Thiadicarbocyanine (DiSC3); Thiazine Red R; Thiazole Orange; Thioflavin 5; Thioflavin S; Thioflavin TON; Thiolyte; Thiozole Orange; Tinopol CBS (Calcofluor White); TIER; TO-PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; TriColor (PE-Cy5); TRITC TetramethylRodaminelsoThioCyanate; True Blue; Tru Red; Ultralite; Uranine B; Uvitex SFC; wt GFP; WW 781; X-Rhodamine; XRITC; Xylene Orange; Y66F; Y66H; Y66W; Yellow GFP; YFP; YO-PRO-1; YO- PRO 3; YOYO- 1; YOYO-3; Sybr Green; Thiazole orange (interchelating dyes); semiconductor nanoparticles such as quantum dots; or caged fluorophore (which can be activated with light or other electromagnetic energy source), or a combination thereof.
In some embodiments, the reporter enzyme includes, but is not limited to luciferase, green fluorescent protein (GFP), yellow fluorescent protein (YFP), blue fluorescent protein (BFP), cyane fluorescent protein (CFP), monomeric red fluorescent protein (mRFP), Discosoma striata (DsRed), mCherry, mOrange, tdTomato, mSTrawberry, mPlum, photoactivatable GFP (PA-GFP), Venus, Kaede, monomeric kusabira orange (mKO), Dronpa, enhanced CFP (ECFP), Emerald, Cyan fluorescent protein for energy transfer (CyPet), super CFP (SCFP), Cerulean, photoswitchable CFP (PS-CFP2), photoactivatable RFP1 (PA-RFP 1), photoactivatable mCherry (PA-mCherry), monomeric teal fluorescent protein (mTFPl), Eos fluorescent protein (EosFP), Dendra, TagBFP, TagRFP, enhanced YFP (EYFP), Topaz, Citrine, yellow fluorescent protein for energy transfer (YPet), super YFP (SYFP), enhanced GFP (EGFP), Superfolder GFP, T-Sapphire, Fucci, mK02, m0range2, mApple, Sirius, Azurite, EBFP, and/or EBFP2.
In some embodiments, the radioactive isotope includes, but is not limited to technetium- 99m, caesium- 137, chromium-51, iodine- 123, palladium- 103, molybdenum-99, iodine-131, iridium- 192, lutetium- 177, copper-64, radium-226, cobalt-60, tritium, americium-241, samarium- 153, and yttrium-90.
The present disclosure also discloses an ND comprising the structure: A-B-C, wherein either A, B, or C comprises a component to direct proteosome degradation of the POI, a component to direct lysosome/autophagosome degradation of the POI, or a component that binds to the POI. In some embodiments, the component directing proteosome degradation comprises a ubiquitin ligand or a ubiquitin ligand complex. In some embodiments, ubiquitin ligand or ubiquitin ligand complex comprises Skpl-Cullin-F-box ubiquitin ligase complex, murine double minute 2 (MDM2) ubiquitin ligase, cereblon (CRBN), cell inhibitor of apoptosis protein (cIAP), and Von- Hipple-Lindau (VHL), including VHL as part of the CRL2VHL E3 ligase complex, complexed with Cullin 4A (CUL4A), DNA-binding protein 1 (DDB1), and ring box protein 1 (RBX1). In some embodiments, the ubiquitin ligand/ubiquitin ligand complex comprises an E3 ligand.
In some embodiments, the component directing lysosome/autophagosome degradation comprises chaperone-mediated autophagy peptides/motifs or chaperone-mediated autophagy protein complex. In some embodiments, the chaperone-mediated peptide/protein or the chaperone- mediated peptide/protein complex comprises B-crystallin, p23, DJ-1, and heat shock protein chaperones, including, but not limited to Hsp90, Hsp70, Hsp27, and Hsp60. In some embodiments, the chaperone-mediated protein/chaperone-mediated protein complex comprises a heat shock protein (Hsp).
In some embodiments, one or more components of any preceding aspect are connected by a linker. In some embodiments, ubiquitin ligand/ubiquitin ligand complex and the chaperone- mediated protein/chaperone-mediated protein complex are linked by a linker. In some embodiments, the ND further comprises moieties including, but not limited to cationicindependent mannose-6-phosphate receptor (CI-MPR aka IGF2FR), poly-M6Pn, asialoglycoprotein receptor (ASGPR), and DNA aptamers Al and A2.
In some embodiments, the ND of any preceding aspect comprises a construct necessary to achieve the desired effect, such as for example targeting the ectodomain, targeting the cytoplasm, and/or targeting the proteasome. In some embodiments, the ND of any preceding aspect comprises an ectodomain-targeting domain construct (Figure 18 A), a covalent binding construct (Figure 18B), a cytoplasmic-targeting nanobody construct (Figure 18C), a bi-paratopic construct (Figure 18D), and/or a proteasome-targeting domain construct (Figure 18E).
Once the concerned sequences have been obtained, the concerned sequences can be obtained in large scale using recombinant methods. Usually, sequences can be obtained by cloning it into a vector, transferring it into cells, and then isolating the sequences from the proliferated host cells by conventional methods. Bio-molecules (nucleic acids, proteins, etc.) to which the present invention relates include bio-molecules that exist in isolated form.
At present, DNA sequences encoding the protein of the present invention (or a fragment thereof, or a derivative thereof) can be obtained completely by chemical synthesis. The DNA sequence then can be introduced into various existing DNA molecules (or e.g., vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.
In one aspect, disclosed herein is a vector comprising a nucleic acid sequence encoding the ND, or a fragment thereof, of any preceding aspect. In some embodiments, the vector is a plasmid, a virus or a viral vector, a phage particle, or a nanoparticle. In some embodiments, the vectors comprise the above-mentioned suitable DNA sequences and suitable promoters or control sequences. These vectors can be used to transform an appropriate host cell so that it can express the protein. In some embodiments, the vector includes, but is not limited to a plasmid derived from pTXBl, pET22, and pET28. In some embodiments, the vector comprises the plasmid of Figure 15. In some embodiments, the vector comprises the plasmid of Figure 16. In some embodiments, the vector comprises the plasmid of Figure 17. In some embodiments, the vector is suitable for integration and/or expression of a fusion protein, such as for example the ND comprising the Nb, the CPP, and the targeting peptide. In some embodiments, the vector comprises a PelB sequence suitable for localizing and/or expressing the ND in the periplasm.
In some embodiments, the vector of any preceding aspect comprises one or more suitable control sequence capable of effecting the expression of the DNA in a suitable host cell. Such control sequences can include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites, and sequences which control the termination of transcription and translation.
The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: Escherichia coli, Slreplomyces. bacterial cells such as Salmonella typhimurium, fungal cells such as yeast, insect cells of Drosophila S2 or Sf9, animal cells of CHO, COST, 293 cells, and the like.
The transformation of the host cell with the recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as E. coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with the CaC12 method. The procedures used are well known in the art. Another method is to use MgC12. If necessary, conversion can also be performed by electroporation. When the host is eukaryotic, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, and the like.
The obtained transformants can be cultured in a conventional manner to express the polypeptide encoded by the gene of the present invention. Depending on the host cells used, the medium used in the culture may be selected from various conventional media. The culture is performed under conditions suitable for the host cells growth. After the host cells are grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature shift or chemical induction) and the cells are incubated for a further period of time.
The recombinant polypeptide in the above method may be expressed intracellularly, or on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods by utilizing its physical, chemical, and other characteristics. These methods are well-known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitation agent (salting out method), centrifugation, osmotic disruption, super treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer analysis, ion exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques and the combinations thereof.
In one aspect, disclosed herein is a kit comprising the ND, the vector, or a salt thereof, of any preceding aspect. In some embodiments, the kit further comprises one or more reagents for stabilizing the ND. In some embodiments, the kit further comprises one or more reagents for delivering the ND to a biological sample or subject. In some embodiments, the kit further comprises one or more reagents for amplifying the vector. In some embodiments, the kit further comprises one or more reagents for expressing the vector in a cell. In some embodiments, the kit further comprises instructions for using the ND, vector, or a salt thereof. In some embodiments, the kit further includes a container, an instruction, a buffer, and the like.
The present disclosure also provides disease-associated neuronal culture models comprising two-layers of co-culturing microglia and neurons derived from isogenic induced pluripotent stem cells (iPSC) lines.
Methods of treating or preventing disease
The present disclosure also provides methods of treating or preventing a disease in a subject in need thereof. The present disclosure also provides methods of decreasing or reducing a disease-related protein.
Herein, a nanodegrader (ND) and methods of use thereof can be applied to any disease affected by or associated with a pathogenic protein. The present disclosure describes a method of administering the ND composition comprising a nanoody (Nb), wherein the ND targets a pathogenic protein.
As used herein, a “pathogenic protein” refers to a fragment or full-length peptide, polypeptide, protein that is produced as a result of a disease or disorder, or is defective or mutated causing the onset or progression of a disease or disorder. The appearance of pathogenic proteins leads to worsening of disease symptoms, addition of more disease symptoms, activation of immunoprotective and/or immunosuppressive signaling pathways, and/or maybe asymptomatic. It should be understood that pathogenic proteins can be endogenous to the subject or arise from an exogenous sources, including, but not limited to the environment, bacteria, virus, fungi, protozoa, and parasites. In some embodiments, the pathogenic proteins are expressed from genetic mutations or disruptions.
In one aspect, disclosed herein is a method of treating or preventing a disease in a subject in need thereof, the method comprises administering a pharmaceutically effective amount of an ND composition comprising a. Nb and a pharmaceutically acceptable carrier, wherein the Nb is operably linked to a cell penetrating peptide (CPP), a targeting peptide, a proximity-enabled crosslinker (PEC), and wherein the ND targets and degrades a disease-related protein, including but not limited to tau, TAR DNA-binding protein 43 (TDP-43), a-synuclein, P-amyloid, a prion protein, human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR), CKLF like MARVEL transmembrane domain-containing 6 (CMTM6), human transcription factor cellular MYC (c-MYC), proliferating cell nuclear antigen (PCNA), or hypoxia-inducible factor 1-alpha (HIF-la).
In one aspect, disclosed herein is a method of treating or preventing a disease in a subject in need thereof, the method comprises administering a pharmaceutically effective amount of the nanodegrader (ND) composition of any preceding aspect.
In some embodiments, the method decreases or reduces the disease-related protein relative to an untreated control. In some embodiments, the methods decreases or reduces the disease- related protein by 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% relative to an untreated control.
In some embodiments, the method further comprises administering the ND and a therapeutic agent. In some embodiments, the therapeutic agent comprises an antibiotic, an antiinflammatory compound, a sedative, an anesthetic, an anti-viral agent, a peptide hormone, an antidiabetic agent, a steroid, or combinations thereof. In some embodiments, the therapeutic agent includes, but is not limited to penicillins (including, but not limited to amoxicillin, clavulanate and amoxicillin, ampicillin, dicloxacillin, oxacillin, and penicillin V potassium), tetracyclins (including, but not limited to demeclocycline, doxycycline, eravacycline, minocycline, omadacycline, sarecycline, and tetracycline), cephalosporins (cefaclor, cefadroxil, cefdinir, cephalexin, cefprozil, cefepime, cefiderocol, cefotaxime, cefotetan, ceftaroline, cefazidme, ceftriaxone, and cefuroxime), quinolones (also referred to as fluoroquinolones include, but are not limited to ciprofloxacin, delafloxacin, levofloxacin, moxifloxacin, and gemifloxacin), lincomycins (including clindamycin and lincomycin), macrolides (including, but not limited to azithromycin, clarithromycin, erythromycin, and fidaxomicin (ketolide)), sulfonamides (including sulfamethoxazole and trimethoprim, and sulfasalazine), glycopeptides (including, but not limited to dalbavancin, oritavancin, telavancin, and vancomycin), aminoglycosides (including, but not limited to gentamicin, tobramycin, and amikacin), carbapenems (including, but not limited to imipenem and cilastatin, meropenem, and ertapenem), and topical antibiotics (including, but not limited to neomycin, bacitracin, polymyxin B, and praxomine), aspirin, ibuprofen, ketoprofen, naproxen, steroids, glucocorticoids (including, but not limited to betamethasone, budesonide, dexamethasone, hydrocortisone, hydrocortisone acetate, methylprednisolone, prednisolone, prednisone, and triamcinolone), methotrexate, sulfasalazine, lefunomide, anti-Tumor Necrosis Factor (TNF) medications, cyclophosphamide, my cophenolate, chloroprocaine, procaine, tetracaine, lidocaine, bupivacaine, ropivacaine, mepivacaine, levobupivacaine, barbiturates, benzodiazepines, nonbenzodiazepines hypnotics, antihistamines, muscle relaxants, opioids, methaqualone, interferons, cytokines (e.g., tumor necrosis factor, interferon a, interferon y), vaccines, hematopoietic growth factors, monoclonal serotherapy, immunostimulants and/or immunodulatory agents (e.g., IL-1, 2, 4, 6, or 12), immune cell growth factors (e.g., GM-CSF) and antibodies (e.g. HERCEPTIN (trastuzumab), T-DM1, AVASTIN (bevacizumab), ERBITUX (cetuximab), VECTIBIX (panitumumab), RITUXAN (rituximab), BEXXAR (tositumomab)), anti-estrogens (e.g. tamoxifen, raloxifene, and megestrol), LHRH agonists (e.g. goscrclin and leuprolide), anti-androgens (e.g. flutamide and bicalutamide), photodynamic therapies (e.g. vertoporfm (BPD-MA), phthalocyanine, photosensitizer Pc4, and demethoxy-hypocrellin A (2BA-2-DMHA)), nitrogen mustards (e.g. cyclophosphamide, ifosfamide, trofosfamide, chlorambucil, estramustine, and melphalan), nitrosoureas (e.g. carmustine (BCNU) and lomustine (CCNU)), alkyl sulphonates (e.g. busulfan and treosulfan), triazenes (e.g. dacarbazine, temozolomide), platinum containing compounds (e.g. cisplatin, carboplatin, oxaliplatin), vinca alkaloids (e.g. vincristine, vinblastine, vindesine, and vinorelbine), taxoids (e.g. paclitaxel or a paclitaxel equivalent such as nanoparticle albumin-bound paclitaxel (ABRAXANE), docosahexaenoic acid bound-paclitaxel (DHA-paclitaxel, Taxoprexin), polyglutamate bound- paclitaxel (PG-paclitaxel, paclitaxel poliglumex, CT-2103, XYOTAX), the tumor-activated prodrug (TAP) ANG1005 (Angiopep-2 bound to three molecules of paclitaxel), paclitaxel -EC- 1 (paclitaxel bound to the erbB2-recognizing peptide EC-1), and glucose-conjugated paclitaxel, e.g., 2'-paclitaxel methyl 2-glucopyranosyl succinate; docetaxel, taxol), epipodophyllins (e.g. etoposide, etoposide phosphate, teniposide, topotecan, 9-aminocamptothecin, camptoirinotecan, irinotecan, crisnatol, mytomycin C), anti-metabolites, DHFR inhibitors (e.g. methotrexate, dichloromethotrexate, trimetrexate, edatrexate), IMP dehydrogenase inhibitors (e.g. mycophenolic acid, tiazofurin, ribavirin, and EICAR), ribonucleotide reductase inhibitors (e.g. hydroxyurea and deferoxamine), uracil analogs (e.g. 5 -fluorouracil (5-FU), floxuridine, doxifluridine, ratitrexed, tegafur-uracil, capecitabine), cytosine analogs (e.g. cytarabine (ara C), cytosine arabinoside, and fludarabine), purine analogs (e.g. mercaptopurine and Thioguanine), Vitamin D3 analogs (e.g. EB 1089, CB 1093, and KH 1060), isoprenylation inhibitors (e.g. lovastatin), dopaminergic neurotoxins (e.g. l-methyl-4-phenylpyridinium ion), cell cycle inhibitors (e.g. staurosporine), actinomycin (e.g. actinomycin D, dactinomycin), bleomycin (e.g. bleomycin A2, bleomycin B2, peplomycin), anthracycline (e.g. daunorubicin, doxorubicin, pegylated liposomal doxorubicin, idarubicin, epirubicin, pirarubicin, zorubicin, mitoxantrone), MDR inhibitors (e.g. verapamil), Ca2+ ATPase inhibitors (e.g. thapsigargin), imatinib, thalidomide, lenalidomide, tyrosine kinase inhibitors (e.g., axitinib (AG013736), bosutinib (SKI- 606), cediranib (RECENTIN™, AZD2171), dasatinib (SPRYCEL®, BMS-354825), erlotinib (TARCEVA®), gefitinib (IRESSA®), imatinib (Gleevec®, CGP57148B, STI-571), lapatinib (TYKERB®, TYVERB®), lestaurtinib (CEP-701), neratinib (HKI-272), nilotinib (TASIGNA®), semaxanib (semaxinib, SU5416), sunitinib (SUTENT®, SU11248), toceranib (PALLADIA®), vandetanib (ZACTIMA®, ZD6474), vatalanib (PTK787, PTK/ZK), trastuzumab (HERCEPTIN®), bevacizumab (AVASTIN®), rituximab (RITUXAN®), cetuximab (ERBITUX®), panitumumab (VECTIBIX®), ranibizumab (Lucentis®), nilotinib (TASIGNA®), sorafenib (NEXAVAR®), everolimus (AFINITOR®), alemtuzumab (CAMPATH®), gemtuzumab ozogamicin (MYLOTARG®), temsirolimus (TORISEL®), ENMD-2076, PCI- 32765, AC220, dovitinib lactate (TKI258, CHIR-258), BIBW 2992 (TOVOK™), SGX523, PF- 04217903, PF-02341066, PF-299804, BMS-777607, ABT-869, MP470, BIBF 1120 (VARGATEF®), AP24534, JNJ-26483327, MGCD265, DCC-2036, BMS-690154, CEP-11981, tivozanib (AV-951), OSI-930, MM-121, XL-184, XL-647, and/or XL228), proteasome inhibitors (e.g., bortezomib (VELCADE)), mTOR inhibitors (e.g., rapamycin, temsirolimus (CCI-779), everolimus (RAD-001), ridaforolimus, AP23573 (Ariad), AZD8055 (AstraZeneca), BEZ235 (Novartis), BGT226 (Norvartis), XL765 (Sanofi Aventis), PF-4691502 (Pfizer), GDC0980 (Genetech), SF1126 (Semafoe) and OSI-027 (OSI)), oblimersen, gemcitabine, caminomycin, leucovorin, pemetrexed, cyclophosphamide, dacarbazine, procarbizine, prednisolone, dexamethasone, campathecin, plicamycin, asparaginase, aminopterin, methopterin, porfiromycin, melphalan, leurosidine, leurosine, chlorambucil, trabectedin, procarbazine, discodermolide, caminomycin, aminopterin, hexamethyl melamine, or derivatives thereof. In some embodiments, one therapeutic agent of any preceding aspect is administered with the ND composition or a nonlimiting number of therapeutic agents of any preceding aspect is administered with the ND composition.
In some embodiments, the disease comprises a neurodegenerative disease, an inflammatory disease, a viral infection, or a cancer. In some embodiments, the disease includes, but is not limited to Alzheimer’s disease, ataxia, Huntington’s disease, Parkinson’s disease, amyotrophic lateral sclerosis (ALS), Frontotemporal dementia (FTD), Friedreich ataxia, Lewy body disease, spinal muscular atrophy, Alpers’ disease, Batten disease, Cerebro-oculo-facio- skeletal syndrome, Leigh syndrome, Prion diseases, monomelic amyotrophy, multiple system atrophy, striatonigral degeneration, motor neuron disease, multiple sclerosis (MS), Creutzfeldt- Jakob disease, Parkinsonism, spinocerebellar ataxia, dementia, acoustic neuroma, adenocarcinoma, adrenal gland cancer, anal cancer, angiosarcoma (e.g., lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma), appendix cancer, benign monoclonal gammopathy, biliary cancer (e.g., cholangiocarcinoma), bladder cancer, breast cancer (e.g., adenocarcinoma of the breast, papillary carcinoma of the breast, mammary cancer, medullary carcinoma of the breast), brain cancer (e.g., meningioma; glioma, e.g., astrocytoma, oligodendroglioma; medulloblastoma), bronchus cancer, carcinoid tumor, cervical cancer (e.g., cervical adenocarcinoma), choriocarcinoma, chordoma, craniopharyngioma, colorectal cancer (e.g., colon cancer, rectal cancer, colorectal adenocarcinoma), epithelial carcinoma, ependymoma, endotheliosarcoma (e.g., Kaposi's sarcoma, multiple idiopathic hemorrhagic sarcoma), endometrial cancer (e.g., uterine cancer, uterine sarcoma), esophageal cancer (e.g., adenocarcinoma of the esophagus, Barrett's adenocarinoma), Ewing's sarcoma, eye cancer (e.g., intraocular melanoma, retinoblastoma), familiar hypereosinophilia, gall bladder cancer, gastric cancer (e.g., stomach adenocarcinoma), gastrointestinal stromal tumor (GIST), head and neck cancer (e.g., head and neck squamous cell carcinoma, oral cancer (e.g., oral squamous cell carcinoma (OSCC), throat cancer (e.g., laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer, oropharyngeal cancer)), hematopoietic cancers (e.g., leukemia such as acute lymphocytic leukemia (ALL) (e.g., B-cell ALL, T-cell ALL), acute myelocytic leukemia (AML) (e.g., B-cell AML, T-cell AML), chronic myelocytic leukemia (CML) (e.g., B-cell CML, T-cell CML), and chronic lymphocytic leukemia (CLL) (e.g., B-cell CLL, T-cell CLL); lymphoma such as Hodgkin lymphoma (HL) (e.g., B-cell HL, T-cell HL) and non-Hodgkin lymphoma (NHL) (e.g., B-cell NHL such as diffuse large cell lymphoma (DLCL) (e.g., diffuse large B-cell lymphoma (DLBCL)), follicular lymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), mantle cell lymphoma (MCL), marginal zone B-cell lymphomas (e.g., mucosa- associated lymphoid tissue (MALT) lymphomas, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma), primary mediastinal B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma (i.e., “Waldenstrom's macroglobulinemia”), hairy cell leukemia (HCL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma and primary central nervous system (CNS) lymphoma; and T-cell NHL such as precursor T-lymphoblastic lymphoma/leukemia, peripheral T-cell lymphoma (PTCL) (e.g., cutaneous T-cell lymphoma (CTCL) (e.g., mycosis fungiodes, Sezary syndrome), angioimmunoblastic T-cell lymphoma, extranodal natural killer T-cell lymphoma, enteropathy type T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, anaplastic large cell lymphoma); a mixture of one or more leukemia/lymphoma as described above; and multiple myeloma (MM)), heavy chain disease (e.g., alpha chain disease, gamma chain disease, mu chain disease), hemangioblastoma, inflammatory myofibroblastic tumors, immunocytic amyloidosis, kidney cancer (e.g., nephroblastoma a.k.a. Wilms' tumor, renal cell carcinoma), liver cancer (e.g., hepatocellular cancer (HCC), malignant hepatoma), lung cancer (e.g., bronchogenic carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung), leiomyosarcoma (LMS), mastocytosis (e.g., systemic mastocytosis), myelodysplastic syndrome (MDS), mesothelioma, myeloproliferative disorder (MPD) (e.g., polycythemia Vera (PV), essential thrombocytosis (ET), agnogenic myeloid metaplasia (AMM) a.k.a. myelofibrosis (MF), chronic idiopathic myelofibrosis, chronic myelocytic leukemia (CML), chronic neutrophilic leukemia (CNL), hypereosinophilic syndrome (HES)), gliomas, neuroblastoma, neurofibroma (e.g., neurofibromatosis (NF) type 1 or type 2, schwannomatosis), neuroendocrine cancer (e.g., gastroenteropancreatic neuroendoctrine tumor (GEP-NET), carcinoid tumor), osteosarcoma, ovarian cancer (e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarian adenocarcinoma), papillary adenocarcinoma, pancreatic cancer (e.g., pancreatic adenocarcinoma, intraductal papillary mucinous neoplasm (IPMN), Islet cell tumors), penile cancer (e.g., Paget's disease of the penis and scrotum), pinealoma, primitive neuroectodermal tumor (PNT), prostate cancer (e.g., prostate adenocarcinoma), rectal cancer, rhabdomyosarcoma, salivary gland cancer, skin cancer (e.g., squamous cell carcinoma (SCC), keratoacanthoma (KA), melanoma, basal cell carcinoma (BCC)), small bowel cancer (e.g., appendix cancer), soft tissue sarcoma (e.g., malignant fibrous histiocytoma (MFH), liposarcoma, malignant peripheral nerve sheath tumor (MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma), sebaceous gland carcinoma, sweat gland carcinoma, synovioma, testicular cancer (e.g., seminoma, testicular embryonal carcinoma), thyroid cancer (e.g., papillary carcinoma of the thyroid, papillary thyroid carcinoma (PTC), medullary thyroid cancer), urethral cancer, vaginal cancer, vulvar cancer (e.g., Paget's disease of the vulva), common cold, influenza ( including, but not limited to human, bovine, avian, porcine, and simian strains of influenza), measles, acquired immune deficiency syndrome/human immunodeficiency virus (AIDS/HIV), anthrax, botulism, cholera, Campylobacter infections, chickenpox, chlamydia infections, cryptosporidosis, dengue fever, diphtheria, hemorrhagic fevers, Escherichia coli (E. coll) infections, ehrlichiosis, gonorrhea, hand-foot-mouth disease, hepatitis A, hepatitis B, hepatitis C, legionellosis, leprosy, leptospirosis, listeriosis, malaria, meningitis, meningococcal disease, mumps, pertussis, polio, pneumococcal disease, paralytic shellfish poisoning, rabies, rocky mountain spotted fever, rubella, salmonella, shigellosis, small pox, syphilis, tetanus, trichinosis (trichinellosis), tuberculosis (TB), typhoid fever, typhus, west nile virus, yellow fever, yersiniosis, zika, coronary artery disease, high/low blood pressure, cardiac arrest/heart failure, congestive heart failure, congenital heart defects/diseases (including, but not limited to atrial septal defects, atrioventricular septal defects, coarctation of the aorta, doubleoutlet right ventricle, d-transposition of the great arteries, Ebstein anomaly, hypoplastic left heart syndrome, and interrupted aortic arch), arrhythmia, peripheral artery disease, stroke, cerebrovascular disease, renal artery stenosis, aortic aneurysm, cardiomyopathies, hypertensive heart disease, pulmonary heart disease, cardiac dysrhythmias, endocarditis, inflammatory cardiomegaly, myocarditis, eosinophilic myocarditis, valvular heart diseases, rheumatic heart diseases, asthma, chronic obstructive pulmonary disease (COPD), pulmonary fibrosis, pneumonia, bronchitis (chronic or acute bronchitis), emphysema, cystic fibrosis/bronchiectasis, pleural effusion, acute chest syndrome, acute respiratory distress syndrome, asbestosis, aspergilosis, severe acute respiratory syndrome (including, but not limited to SARS-CoV-1 and SARS-CoV- 2), respiratory syncytial virus (RSV), middle eastern respiratory syndrome (MERS), mesothelioma, pneumothorax, pulmonary arterial hypertension, pulmonary hypertension, pulmonary embolism, sarcoidosis, sleep apnea, albinism, amniotic band syndrome, anencephaly, Angelman syndrome, Barth syndrome, chromosomal abnormalities (including, but not limited to abnormalities to chromosome 9, 10, 16, 18, 20, 21, 22, X chromosome, and Y chromosome), cleft lip/palate, club foot, congenital adrenal hyperplasia, congenital hyperinsulinism, congenital sucrase-isomaltase deficiency (CSID), cystic fibrosis, De Lange syndrome, fetal alcohol syndrome, first arch syndrome, gestational diabetes, Haemophilia, heterochromia, Jacobsen syndrome, Katz syndrome, Klinefelter syndrome, Kabuki syndrome, Kyphosis, Larsen syndrome, Laurence-Moon syndrome, macrocephaly, Marfan syndrome, microcephaly, Nager’s syndrome, neonatal jaundice, neurofibromatosis, Noonan syndrome, Pallister-Killian syndrome, Pierre Robin syndrome, Poland syndrome, Prader-Willi syndrome, Rett syndrome, sickle cell disease, Smith- Lemli-Optiz syndrome, spina bifida, congenital syphilis, teratoma, Treacher Collins syndrome, Turner syndrome, Umbilical hernia, Usher syndrome, Waardenburg syndrome, Werner syndrome, Wolf-Hirschhom syndrome, Wolff-Parkinson-White syndrome, heartbum, irritable bowel syndrome, lactose intolerance, gallstones, cholecystitis, cholangitis, anal fissure, hemorrhoids, proctitis, colon polyps, infective colitis, ulcerative colitis, ischemic colitis, Crohn’s disease, radiation colitis, celiac disease, diarrhea (chronic or acute), constipation (chronic or acute), diverticulosis, diverticulitis, acid reflux (gastroesophageal reflux (GER) or gastroesophageal reflux disease (GERD)), Hirschsprung disease, abdominal adhesions, achalasia, acute hepatic porphyria (AHP), anal fistulas, bowel incontinence, centrally mediated abdominal pain syndrome (CAPS), clostridioides difficile infection, cyclic vomiting syndrome (CVS), dyspepsia, eosinophilic gastroenteritis, globus, inflammatory bowel disease, malabsorption, scleroderma, volvulus, diabetes mellitus Type I, diabetes mellitus Type II, familial hypercholesterolemia, Gaucher disease, Hunter syndrome, Krabbe syndrome, metachromatic leukodystrophy, Niemann- Pick syndrome, phenylketonuria (PKU), Tay-Sachs disease, Wilson’s disease, hemachromatosis, mitochondrial disorders or diseases (including, but not limited to Alpers Disease; Barth syndrome; beta. -oxidation defects :carnitine-acyl-camitine deficiency; carnitine deficiency; coenzyme Q10 deficiency; Complex I deficiency; Complex II deficiency; Complex III deficiency; Complex IV deficiency: Complex V deficiency; cytochrome c oxidase (COX) deficiency, LHON Leber Hereditary Optic Neuropathy; MM Mitochondrial Myopathy: LIMM Lethal Infantile Mitochondrial Myopathy; MMC Maternal Myopathy and Cardiomyopathy; NARP Neurogenic muscle weakness, Ataxia, and Retinitis Pigmentosa; Leigh Disease: FICP — Fatal Infantile Cardiomyopathy Plus, a MELAS-associated cardiomyopathy: MELAS Mitochondrial Encephalomyopathy with Lactic Acidosis and Stroke-like episodes; LDYT Leber's hereditary optic neuropathy and Dystonia; MERRF Myoclonic Epilepsy and Ragged Red Muscle Fibers; MHCM Maternally inherited Hypertrophic CardioMyopathy; CPEO Chronic Progressive External Opthalmoplegia; KSS Kearns Sayre Syndrome; DM Diabetes Mellitus; DMDF Diabetes Mellitus+DeaFness; CIPO Chronic Intestinal Pseudoobstruction with myopathy and Opthalmoplegia; DEAF Maternally inherited DEAFness or aminoglycoside-induced DEAFness; PEM Progressive encephalopathy; SNHL SensoriNeural Hearing Loss; Encephalomyopathy; Mitochondrial cytopathy: Dilated Cardiomyopathy: GER Gastrointestinal Reflux: DEMCHO Dementia and Chorea; AMDF Ataxia, Myoclonus; Exercise Intolerance: ESOC Epilepsy, Strokes, Optic atrophy, & Cognitive decline; FBSN Familial Bilateral Striatal Necrosis: FSGS Focal Segmental Glomerulosclerosis: LIMM Lethal Infantile Mitochondrial Myopathy; MDM Myopathy and Diabetes Mellitus: MEPR Myoclonic Epilepsy and Psychomotor Regression; MERME MERRF/MELAS overlap disease; MHCM Maternally Inherited Hypertrophic CardioMyopathy; MICM Maternally Inherited Cardiomyopathy; MILS Maternally Inherited Leigh Syndrome; Mitochondrial Encephalocardiomyopathy; Multisystem Mitochondrial Disorder (myopathy, encephalopathy, blindness, hearing loss, peripheral neuropathy); NAION Nonarteritic Anterior Ischemic Optic Neuropathy; NIDDM Non-Insulin Dependent Diabetes Mellitus; PEM Progressive Encephalopathy; PME Progressive Myoclonus Epilepsy; RTT Rett Syndrome: SIDS Sudden Infant Death Syndrome: MIDD Maternally Inherited Diabetes and Deafness; and MODY Maturity-Onset Diabetes of the Young, and MNGIE), alcoholic cardiomyopathy, systemic carnitine deficiency, malonyl carboxylase deficiency, malonic aciduria, camitine-acylcarnitine translocase deficiency, carnitine palmitoyltransferase II deficiency, deficiencies to mitochondrial beta-oxidation (including, but not limited to medium-chain acyl-coenzyme A (coA) dehydrogenase (MCAD) deficiency, short-chain acyl-coA dehydrogenase (SCAD) deficiency, very-long-chain acyl-coA dehydrogenase (VLCAD) deficiency, and long-chain 3-hydroxyacyl- coA dehydrogenase (LCHAD) deficiency), deficiencies to the mitochondrial electron respiratory chain (including, but not limited to Kearns- Sayre syndrome, MEL AS syndrome, MERRF syndrome, Barth syndrome, Leigh’s syndrome, Pearson syndrome, respiratory chain complex I deficiency, and Complex III deficiency), Glycogen storage disease type II (Pompe disease), Glycogen storage disease type III, Niemann-Pick disease, Gaucher disease, Lcell disease, mucopolysaccharidosis type I (Hurler syndrome), mucopolysaccharidosis type II (Hunter syndrome), mucopolysaccharidosis type III (Harris- Sanfilippo syndrome), mucopolysaccharidosis type IV (Morquio syndrome), mucopolysaccharidosis type VI (Maroteaux-Lamy syndrome), GM1 gangliosidosis, galactosialidosis, carbohydrate deficient glycoprotein syndromes, Sandhoff s disease, congenital heart defects, and other diseases.
The ND composition may be administered in such amounts, time, and route deemed necessary in order to achieve the desired result. The exact amount of the ND composition will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular ND composition, its mode of administration, its mode of activity, and the like. The ND composition is preferably formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the ND composition will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disease being treated and the severity of the disease symptoms; the activity of the ND composition employed; the specific ND composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific ND composition employed; the duration of the treatment; drugs used in combination or coincidental with the ND composition employed; and like factors well known in the medical arts.
The ND composition may be administered by any route. In some embodiments, the ND composition is administered via a variety of routes, including oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, mucosal, nasal, buccal, enteral, sublingual; by intratracheal instillation, bronchial instillation, and/or inhalation; and/or as an oral spray, nasal spray, and/or aerosol. In general, the most appropriate route of administration will depend upon a variety of factors including the nature of the ND composition (e.g., its stability in the environment of the subject’s body), the condition of the subject (e.g., whether the subject is able to tolerate oral administration), etc.
The exact amount of ND composition required to achieve a therapeutically or prophylactically effective amount will vary from subject to subject, depending on species, age, and general condition of a subject, severity of the side effects, identity of the particular compound(s), mode of administration, and the like. The amount to be administered to, for example, a child or an adolescent can be determined by a medical practitioner or person skilled in the art and can be lower or the same as that administered to an adult.
In one aspect, disclosed herein is a nanodegrader composition comprising a nanobody of any preceding aspect and a pharmaceutically acceptable carrier selected from an excipient, a diluent, a salt, a buffer, a stabilizer, a lipid, an emulsion, a nanoparticle, and a cream. One or more active agents (e.g. the nanobody, the CPP, and the targeting peptide) can be administered in the “native” form or, if desired in the form of salts, esters, amides, prodrugs, or a derivative that is pharmacologically suitable. Salts, esters, amides, prodrugs, and other derivatives of the active agents can be prepared using standards procedures known to those skilled in the art of synthetic organic chemistry and described, for example, by March (1992) Advanced Organic Chemistry; Reactions, Mechanisms, and Structure, 4th Ed. N.Y. Wiley -Interscience.
In some embodiments, the ND composition can be prepared as a “concentrate”, e.g. in a storage container of a premeasure volume and/or a predetermined amount ready for dilution, or in a soluble capsule ready for addition to a specified volume of water, saline, alcohol, hydrogen peroxide, or other diluent.
In some embodiments, the ND composition is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more times. In some embodiments, the ND composition is administered daily. In some embodiments, the ND composition is administered weekly. In some embodiments, the ND composition is administered monthly. In some embodiments, the ND composition is administered yearly.
In one aspect, disclosed herein is a method of degrading or eliminating a disease-related protein in a subject or biological sample, the method comprising introducing a nanodegrader (ND) composition to the subject or biological sample, wherein the ND comprises a chimeric nanobody (Nb) and pharmaceutically acceptable carrier, wherein the Nb is operably linked to a cell penetrating peptide (CPP), a targeting peptide, and a proximity-enabled crosslinker (PEC), internalizing the ND into a cell of the subject or biological sample, wherein the CPP directs internalization into the cell, irreversibly targeting and binding the ND to the disease-related protein, wherein the PEC binds the disease-related protein to the ND, and degrading the disease- related protein, wherein the targeting peptide activates at least one proteolytic pathway to decrease the disease-related protein relative to a control subject or biological sample.
In one aspect, disclosed herein is a method of degrading or eliminating a disease-related protein in a subject or biological sample, the method comprising introducing a nanodegrader (ND) composition of any preceding aspect to the subject or biological sample, internalizing the ND into a cell of the subject or biological sample, wherein the CPP directs internalization into the cell, irreversibly targeting and binding the ND to the disease-related protein, wherein the PEC binds the disease-related protein to the ND, and degrading the disease-related protein, wherein the targeting peptide activates at least one proteolytic pathway to decrease the disease-related protein relative to a control subject or biological sample.
Proteolysis refers to the cellular process of shedding/degrading proteins into smaller units such as, for example polypeptides, amino acids, carbon molecules, nitrogen molecules, and/or hydrogen molecules. Proteolysis in mammalian cells occurs by several mechanisms including a ubiquitin-proteosome pathway and/or a lysosomal proteolytic pathway. Ubiquitin-proteosomal proteolysis occurs by the following steps: (1) covalent attachment of at least one ubiquitin molecule to a targeted protein, such as for example a misfolded protein and/or a dysfunctional protein; and (2) degradation of the targeted protein by the 26S proteosome complex with the release of free and reusable ubiquitin. E3 ligase is the enzyme responsible for attaching the ubiquitin to the targeted protein(s). Herein, the Nb is conjugated with E3 ligase ligands to catalyze the attachment of ubiquitin to targeted proteins. Ishida et al. is incorporated by reference herein for its teachings regarding discovery and descriptions of E3 ligase ligands for PROTACs (Ishida et al. “E3 Ligase Ligands for PROTACS: How They Were Found and How to Discover New Ones” SLAS Discov. April 2021; 26(4): 484-502).
Proteolysis also occurs by lysosomal degradation wherein targeted proteins, such as for example misfolded proteins and/or dysfunctional proteins, are trafficked into cellular vesicles, called lysosomes, for degradation. Lysosomes are acidic, membrane-bound cytoplasmic organelles found within various types of cells that can degrade various biomolecules, including proteins. Trafficking of targeted proteins into lysosomes occurs by macroautophagy, chaperone- mediated autophagy, endocytosis, or pinocytosis. Jackson et al. is incorporated by reference herein for its teachings of degrading misfolded proteins by lysosomes (Jackson et al. “Cellular proteostasis: Degradation of misfolded proteins by lysosomes” Essays Biochem. October 15, 2016; 60(2): 173-180).
In some embodiments, the method of any preceding aspect comprises a Nb comprising SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, or a variant thereof In some embodiments, the method of any preceding aspect comprises a ND comprising a CPP with SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, or a variant thereof
In some embodiments, the at least one proteolytic pathway comprises a ubiquitin- proteosome pathway, a lysosomal proteolytic pathway, or a combination thereof.
In some embodiments, the method treats or prevents a disease of any preceding aspect.
In some embodiments, the biological sample comprises a tissue biopsy, a cerebrospinal fluid (CSF) sample, a blood sample, a serum sample, or an isolated cell. In some embodiments, the biological sample is in vitro or in vivo. In some embodiments, the isolated cell is a neuron, a cancer cell, or an immune cell (including, but not limited to a B cell, T cell, and natural killer (NK) cell).
The present disclosure also provides methods to identify potential therapeutic compositions for diseases, including but not limited to Parkinson’s disease, comprising human neuronal co-culturing a therapeutic composition with neurons with a-synuclein (a-syn) A35T mutations or neurons treated with recombinant a-syn short fibrils, and identifying therapeutic compositions.
In addition to protein misfolding diseases, the present invention seeks to advance the therapeutic landscape for cancers by targeting but not limited to the selective degradation of the aggressive forms of the oncogenes: EGFR, HER2, CMTM6, cMYC, PCNA, HIF-la.
A number of embodiments of the disclosure have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.
By way of non-limiting illustration, examples of certain embodiments of the present disclosure are given below. EXAMPLES
The following examples are set forth below to illustrate the compositions, devices, methods, and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention which are apparent to one skilled in the art.
Example 1: Nanobody-based Protein Degraders and Related Methods
Targeted protein degradation (TPD) strategies exemplified by proteolysis targeting chimeras (PROTACs) are desirable to overcome inherent off-target effects from genetic strategies CRISPR/Cas9 and RNA interference. PROTAC employs the ubiquitin-proteasome system (UPS), a major intracellular protein degradation system, to degrade a protein of interest (POI) (Figure 7 A). However, this method has been only applied to targets with available small molecule inhibitors, and classically “undruggable” proteins remain challenging. Particularly, this strategy also proves unworkable for targeting misfolded proteins in neurodegenerative diseases. Recent advances in “nanobody”, the smallest-known functional antibody fragment (<15 kDa) that is able to be delivered into living cells, have demonstrated its significant translational functionality in both preclinical and clinical studies. Several intracellular nanobody-based TPD approaches have been developed but they require a fusion of nanobody with an E3 ligase catalytic domain or a subunit of the E3 ligase complex that causes unexpected cell toxicity. To address the above- mentioned limitations, a method exploiting the efficiency of nanobody and PROTAC is developed to approach close together POI and proteasome machinery to stimulate rapid POI degradation: nano-degraders.
The present disclosure provides the development of an intein-mediated expressed protein ligation (EPL) to site- selectively conjugate a nanobody (Nb) with small E3 ligase selective ligands. The ALFA tag-specific nanobody was used for developing EPL approach. A 25-mer peptide (Nb aa 98-122) was ligated to the intein-mediated Nb (aal-97) thioester fragment to produce full length ALFA Nb that was shown >95% ligation yield (Figure 7C). It has been contemplated that the E3 ligase complex can attach ubiquitin to the Nb Lysine residues and drives its proteasomal degradation quickly. To avoid this, non-Lysine ALFA Nb variant was generated and shown functional as the wild type (Figure 7D). It was also planned to introduce proteaseresistant D-amino acids into Nb via the EPL strategy to enhance its half-life. The established EPL is used to generate nano-degraders specific to misfolded TDP-43 or Tau. A crucial step for the success of this method is the delivery of the nano-degraders into the cells. The nano-degraders are decorated with a highly efficient cyclic D-Arginine-rich CPP12 (Figure 7B) that was proved to deliver Nbs into live cells and mice (See, Zhang, W., et al., An intracellular nanobody targeting T4SS effector inhibits Ehrlichia infection. Proc Natl Acad Sci U S A, 2021. 118(18)). Recently, a small (<10 kDa) membrane translocation domain (MTD) was delivered as an efficient CPP to deliver a target protein for the degradation of oncoprotein K-Ras in cancer cells. It has been further contemplated that nanodegraders (NDs) can also be fused with MTD as an alternative strategy.
Example 2: Development of nanobody based protein degraders targeting misfolded proteins in neurodegenerative disease.
Neurodegenerative diseases including Alzheimer's and Parkinson’s diseases, amyotrophic lateral sclerosis (ALS), prion diseases, and among others affect millions of people in the US. Unfortunately, there is currently no adequate treatment for these diseases. One of the earliest steps in the triggers of these diseases is the misfolding, aggregation, and accumulation of proteins in the central nervous system (CNS). Thus, selective elimination of misfolded but not normal version of proteins is a therapeutic strategy to mitigate the diseases. Here, a novel protein degrader is developed targeting misfolded proteins in human CNS cell models for neurodegenerative disorders.
Processing of misfolded proteins is important in order for the cell to maintain its normal functioning and homeostasis. In particular, it is noted herein that ALS and tauopathies with two of the most common spectrum of TAR-DNA binding protein 43 (TDP-43) and Tau pathologies in neurodegenerative diseases, are characterized by altered protein clearance and deposits. Thus, the manipulations of proteasome systems are useful in these and other aggregation-related diseases. The present disclosure uses nano-degraders to target TDP-43 and Tau aggregates. The human CNS cells differentiated from induced pluripotent stem cells (iPSCs) models is used to validate the nano-degrader method by analyzing its effects on the aggregate clearance and alleviate cellular pathological feature of human CNS cells. This invention yields promising chemical biology strategies to counteract protein aggregates-induced cellular toxicity not only in ALS and Tauopathies but other misfolded protein disorders.
The present disclosure also provides human iPSCs systems with either ALS -associated TDP-43 Q33 IK mutant, ALS/FTD-linked Ubiquilin2 P497H/P506T mutant or Parkinson diseaselink a-synuclein A53T mutant differentiated into CNS cell types including upper or lower motor neurons, microglia, and astrocytes using our established protocol (Figure 1 and Figure 8A). The expression, biochemical properties, cellular localization of protein aggregates are analyzed in iPSC-derived cells (Figure 9 and Figure 10). The effects on cellular clearance of aggregated forms induced by nano-degraders are characterized in these human cells. It is also tested whether this method ameliorates the cellular pathological features including the abnormalities in morphology and function of human CNS cell with these disease-associated mutations (Figure 11).
The ability to induce protein degradation in a directed manner by nano-degrader allows for the extraction of important information from biological systems, especially for the undruggable proteome and specific conformation of targets. Moreover, this technology is emerging as a highly versatile tool since nanobodies against every POI can be easily modified to become nanodegraders. In addition, the application of iPSCs models is significantly extended to the studies of the realm of human CNS cells.
1. Neurodegenerative disease human models
Herein are specialized culture systems with neurons carrying disease-associated mutations to successfully recapitulate the corresponding diseases with several key pathological features, including the presence of pathological protein aggregates (Figure 2) together with the induction of synaptic abnormalities (Figure 3). The system provides powerful models for mechanistic studies and testing the efficacy of therapeutic molecules that are directly relevant to human diseases, where defined pathological abnormalities in physiological conditions of expressing endogenous proteins. a. Protein pathology
Protein pathology is a hallmark of neurodegenerative diseases including PD, ALS and prion diseases and causes neuronal dysfunction and death. Tauopathies, a-synucleinopathies, and TDP43 proteinopathies are some of the most common neurodegenerative disorders. Mutations in disease-associated genes such as TDP43 (TARDBPQ331K, Figure 2A) and Ubiquilin2 (UBQLN2P497H/P506T, Figure 2B), and a-syn (SNCAA53T, Figure 2C) have been identified to cause familial forms of ALS, ALS/FTD and PD, respectively. The mutations cause pathological aggregates and disrupt the ability of cellular machinery to clear the misfolded proteins. In prion diseases, one of the most recognized human prion disorders is Creutzfeldt-Jakob disease (CJD). Cellular prion protein (PrPC) with E200K mutation associated with inherited CJD PBNPE200K, Figure 2D), which exemplifies fundamental biological principles now known to apply to a wide range of human neuropathology. The neurotoxicity caused by prions (PrPSc), the misfolded form of PrPC, is related to the activation of specific signal transduction pathways. We analyzed the protein pathology in neurons carrying those disease-associated mutations. Immunoassays of mature neurons indicated increased protein levels and aggregation of disease-hallmark proteins, including TDP43, a-syn, UBQLN2, prion protein, and some pathological phosphor-forms in mutant lines (Figure 2). Mouse models and patient tissues showed increased levels of phospho tau proteins which co-localized to misfolded protein and deposited in the protein inclusions. Visualizing misfolded proteins by immunofluorescence (IF) display accumulates and co-localizes with paired helix filament (PHF) Tau in mutant neurons within the soma and dendritic regions (Figures 2A, Cl and D2). b. Synaptotoxicity
Synapse loss in neurodegeneration is caused by protein aggregate formation. Synaptic abnormalities can be observed before diagnosis in certain patients with the diseases, showing that protracted preclinical structure/function alterations play a key role in disease pathogenesis. Some membrane channel subunits may be retained intracellularly, as a result of their misfolded forms being affected by the mutations. This may cause abnormalities in synaptic protein composition, trafficking and localization, and lead to alterations in synaptic morphology and/or connectivity. The model system allows for access to the neurons at the level of synapses. Visualizing spine morphology, a significant reduction of dendritic spine number was observed in mutant neurons (Figure 3). These data show that mutation may exert an effect on synaptic elements.
2. Nanobody-based chemical biology tools.
Nanobody (Nb), the smallest-known functional antibody fragment, has demonstrated its significant translational potential in both preclinical and clinical studies. Herein, chemical biology tools (Figure 1A) were developed for targeting misfolded proteins, named nano-crosslinkers (NCs) and nano-degraders (NDs), to study protein interactomes and networks, and therapeutically eliminate them in human disease neuronal conditions. The expressed protein ligation (EPL) method was established and these tools were designed utilizing three major technologies. First, Nbs are used to replace conventional antibodies to facilitate cell penetration. Second, the engineered Nb chimeras irreversibly react with the protein of interest (POI) by covalent interaction to overcome relatively low binding affinity and minimize off-target effects. Third, a cellpenetrating peptide (CPP), and targeting sequences such as chaperon-mediated autophagy (CMA) peptide, macrocyclic (MC) peptides, E3 ligands are conjugated to the Nbs to promote the internalization and targeted degradation. This strategy has been successfully tested with the artificial ALFA tag Nb (NbALFA) by creating the NCALFA (Figures 4A and 4B) and NDALFA (Figures 4C, 4D, 4E, and 4F) that target ALFA tagged-EFGP endogenously expressed in Hela cells and human neurons.
Nb-based Proximity Crosslinkers (NCs): By adding a proximity-enabled covalent bonding of Nbs with POIs to improve their on-target retention, thereby enhancing the spatial resolution level of other chemical species such as photocrosslinker diazirine incorporated into the Nbs. Proximity enabled sulfur-fluoride exchange (SuFEx) covalent linking is a suitable chemistry since the fluorosulfate warhead is biocompatible and able to react with multiple natural residues under physiological conditions (Figure 4A). The EPL strategy was used to incorporate fluorosulfate-containing amino acids (FS-AAs) into NbALFA, which revealed a proximity-enabled X-linking with ALFA-tagged proteins. The NCALFA is successfully internalized into neurons, then targeted and cross-linked with expressed ALFA-tagged EGFP in live neurons. Remarkably, the X-linking product of NCALFA and ALFA-tagged EGFP was successfully isolated by co-IP (Figure 4B) in live neurons, this result proves the promising advantage of this method in studying protein complexes and PPIs. In addition to EPL strategy, the click chemistry strategy was used to incorporate FS-AAs into the Nb N-terminus (Fig 12).
Nb-based Degraders (NDs): ALFA tag nanodegrader (NDALFA) was successfully developed (Figures 4C, 4D, 4E, and 4F) that induced a complete degradation of ALFA-EGFP in Hela (Figures 5A, 5B, and 5C) and human neurons (Figures 5D, 5E, and 5F). In addition, the specific targeting of NDALFA was observed at the dendritic spine locations of neurons (Figure 5D, right panels) which is an advantage for targeting supersaturated/misfolded synapse proteins. Overall, the tunable Nb-based systems developed herein enable targeted monitoring of how protein regulation drives cellular signaling. To obtain dual proteolytic signals for both proteasome and lysosome systems that prove a highly efficient clearance of misfolded proteins, the proteasome-targeting motifs such as peptide sequences (i.e. targeting VHL, Mdm2 E3 ligases) or E3 ligase-specific small molecules (Cereblon, VHL, IAPS, Mdm2, etc. ...) will be incorporated into CMA-contained NDs by recombinant DNA cloning or protein chemical approaches such as EPL (Figures 4 and 7) and click chemistry (Figure 13).
3. Application. a. Targeting pathological a-syn in Parkinson ’s disease models
Building on data using the NDALFA, a NDs“'syn was also developed which has successfully targeted and induced degradation of WT protein and A53T a-syn aggregates in Hela cells (Figures 5G, 5H, and 51). Dual proteolytic signals for both proteasome and lysosome systems were created to prove a highly efficient clearance of misfolded proteins, by additional conjugation with a proteasome directing agents (Pro) such as E3 ligand NHS esters using a stoichiometry chemical approach (as shown in Figure 4F). Protease-resistant D-amino acids were introduced intoNb via the N-terminal EPL strategy to enhance the tools half-life. The rescue efficacy of NDs was investigated in mutant neurons based on their effects on the clearance of target proteins as well as rescuing protein pathology (Figure 2) and synaptotoxicity (Figure 3), including abnormal levels of synaptic proteins, synaptic transmission, and synaptic morphologies. b. Targeting PrP in ALS/FTD-linked UBQLN2 models
The physiological cellular form PrPC is present on the outside leaflet of the membrane of most cell types. In neurons, PrPC is predominant in axons and dendrites. PrPC involves several cellular processes, including neuritogenesis, neuronal homeostasis, cell signaling, cell adhesion, and a protective role against stress. PrPC serves as cellular receptors for b-sheet-rich neurotoxic proteins including a-syn and TDP43. UBQLN2 protein structure contains four stress-induced protein 1 (STI-l)-like domains, which are involved in the interaction with heat shock proteins and autophagy mediators. Mutation in these domains also causes ALS. STI1 protein is found as a cell surface ligand for PrPC and their interaction triggers neuroprotection. The sagittal brain sections of two lines of mice, which express UBQLN2WT or UBQLN2P497S, were stained. Staining revealed the presence of numerous PrP inclusions, which are colocalized with UBQLN2+ aggregates in end-stage P497S animals. The inclusions were rarely seen in the brains of equivalent age UBQLN2WT expressing lines where instead PrP staining was more uniform. Similarly, the staining of spinal cord (Sc) sections contained large irregularshaped structures in the gray matter that immunoreactivity with PrP antibodies. PrP staining is positive to Ubiquitin. Some UBQLN2 inclusions, which are positive to PrP, are also found colocalized with aggregated TDP43. Data shows that PrP constitutes ubiquitinated proteins with amyloid conformations. Then, the effect on PrPC degradation under the expression of UBQLN2P497H was analyzed in the N2a cell line, an overexpressed PrP cell line. A delay in the degradation rate of PrPC affected by the P497H mutation was observed. Co-immunoprecipitation assay of PrP and UBQLN2 proteins in Hela shows intermolecular interaction between PrP and mutant UBQLN2 proteins. More important, cells and neurons with UBQLN2 mutation increase the appearance of cytoplasmic PrP. Any event leading to the accumulation of PrP in the cytoplasm results in cellular toxicity. Agreeing with data from transgenic mice, a consistent aggregation of PrPC was found, which is positive to amyloid dye Thioflavin S in mutant neurons. The data on mouse and human neuron models with UBQLN2 mutations show a novel pathological role of PrPC link to ALS-UBQLN2. Even though PrPC is a membrane-anchored protein, the reasons why PrP was accumulated in the cytoplasm with UBQLN2 and TDP43 inclusions are unknown. To answer this question, the interactomes of PrPC, including its functional form at the membrane (Figure 6C) and the abnormal cytoplasmic forms (Figure 6D) were studied. The conventional proximity biotin-labeling methods do not provide direct information about PPIs. Other antibody -based mapping techniques such as pMap have only been applied for cell membrane surfaces since it is challenging to deliver such large antibodies into the cells. Alternatively, more controllable photo-crosslinking reactions also have been used to capture the physical interactome. However, the incorporation of photo-X-linkers into target proteins using amber codon suppression remains a challenge due to cytotoxicity from proteome- wide nonspecific incorporation. In addition, genetically encoded tags such as Halo, Snap and others only recruit a single photo-X-linker to target protein, therefore limiting their capturing ability. These limitations are addressed herein by efforts to develop proximity crosslinking tools, NCPrP, for use in capturing PrP protein complexes of UBQLN2 mutant neurons (Figure 6). Data with NCALFA (Figure 4B) to make the NCPrP and investigate which protein partners and networks that are related to PrPC-mediated ALS-UBQLN2. The ability to create photo-activated protein X- linkers in a directed manner by Nbs and in a high spatial resolution level assisted by SuFEx X- linking allows for extraction of important biological information for understanding how PrPC and its partners are specifically involved in mutant UBQLN2-mediated neurotoxicity. This data enlightens the understanding of PrP's function as a receptor in ALS-UBQLN2 that mediates misfolded protein neurotoxic effects, such as misfolded TDP43 and mutant UBQLN2 proteins. The proximity-enabled X-linking processes is followed by enrichment purification, tryptic digestion and MS/MS identification of the PrP interactomes (Fig 6B). To further validate the enriched subset of proteins identified with NCPrP, targeted labeling of the highly enriched proteins is performed herein. Targeted NCPrP of these proteins within the PrPC microenvironment, therefore, affords similar enrichment lists, verifying their spatial association. c. Targeting pathological TDP-43 in ALS disease models
NDs targeting TDP43 aggregates (NDTDP43) have been developed using the scFV domains derived from heavy chain (VH) and light chain (VL) of the anti-TDP43 aggregate antiboby 3B12A. Thus far, multi-milligrams of anti-TDP-43 VH domain in fusion with lysosome-targeting CMA1 tag have been purified from 1 L of E. coli expression. For the anti-TDP43 VL domain, an N-terminal fusion with the VH domain and the E. coli periplasmic signal peptide PelB was proved to be helpful in enhancing the solubility of the VL domain. A similar protein chemical approach described above has been used to make NDSTDP43 from purified VH and VL domains.
Example 3: Targeted degradation of oncogenic EGFR and HER2 using nanobodies as proximity-directing agents.
About 40% of encoding genes produce proteins that are either extracellular or membranebound or associated. Traditional inhibitory methods using either small molecules or biologies (i.e. moclonal antibodies), often struggle with these targets owing to their multifunctional roles and complex structures. Promising biological PROTACs (bioPROTACs) have been developed that successfully target extracellular and membrane proteins. However, bioPROTACs, including antibody-based PROTAC (AbTAC) and lysosomal-targeting chimeras (LYTACs), encounter challenges. These include dependence on lysosomal-targeting receptors (LTRs), large antibodies scaffold, and membrane E3 ligases that may be downregulated or subjected to loss-of-function mutations in certain cancers. Moreover, the presence of the Fc region in LYTACs and the intricacy of attaching glycans can impact immune responses and pose challenges in manufacturing and scalability. Many existing bioPROTACs work via intracellular overexpression, limiting them to the cytoplasm, and can hinder target accessibility, increase cytotoxicity, reduce engineering control, and prevent modulation of the tumor environment or impact multiple cells. Here, we address these limitations by developing nano-degraders targeting some key oncogenic membrane proteins such as EGFR and HER2. In addition, we also want to draw an attention to CKLF like MARVEL transmembrane domain-containing 6 (CMTM6) overexpression that has been proposed to confer a resistance to the FDA-approved immunotherapy using trastuzumab antibody in HER2- positive breast cancers.
SKBR3, MDA-MB-231, MCF7, and HEPG2 cell lines will be used to evaluate the delivery efficiency and degradation potency of NDs. Luciferase complementation assay, NanoLuc, will be employed to determine the delivery efficiency and endosomal escape of the NDs. It will be evaluated by fusing HiBit, an 11 -ami no acid peptide from NanoLuc. This involves transfecting the cells with plasmid DNA encoding LgBiT, an 18 kDa subunit of NanoLuc. Successful cytosolic delivery and escape results in HiBit binding to LgBiT, forming an active luciferase, NanoLuc. The degradation activity would be analyzed following NDs incubation for 24 hours in varying doses. First, the relative surface expression of the targets by flow cytometry using an orthogonal detection antibody against the targets. This experiment will directly quantify cell surface expression of EGFR and HER2 after ND treatment. Western blot and confocal imaging with validated antibodies will be used to determine the total degradation activity of NDs. Numeric values obtained from the western blot and confocal imaging quantification will be used to create the dose-response curve for target proteins degradation to determine the half-maximal degradation concentration (DC50) and the maximum level of target protein degradation (DraaK). The time course of degradation of EGFR and HER2 in the cells will be carried out for 2 - 48 h using the optimal dose. The use of immunoblot will contribute to assessing the impact of ND treatment on the levels of downstream effectors, specifically phosphorylated form of pAkt and pERKl/2. Quantitative mass spectrometry by Tandem Mass Tag will be employed to confirm the specificity of degradation across the proteome under NDs treatment. Specific 26S proteasome inhibitors, such as MG132 or Bortezomib, would be used to assess if clearance by NDs is via the proteasome. Lastly, we will employ specific assays following manufacturer protocols to evaluate their antiproliferative, migration effect (Incucyte S3 Live-Cell Analysis System) and cell viability by luminometer (CellTiter-Glo Luminescent Cell Viability reagent). We will use transwell migration assays to assess cell migration. Cells will be seeded on a porous membrane dividing two chambers, using 10% FBS in the lower chamber as a chemoattractant. After incubation, non-migrated cells will be cleared, and migrated cells on the membrane's bottom side will be fixed with 4% PF A, stained, and quantified to assess migration.
It will be apparent to those skilled in the art that various modifications and variations can be made in the present disclosure without departing from the scope or spirit of the invention. Other embodiments of the disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the methods disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.
SEQUENCES
1. SEQ ID NO: 1 - a-Synuclein Nanobody (Nb) (Reference: PMID: 23557833)
MSGGSENLYFQCGGYPYDVPDYASGGQVQLQESGGGSVQTGGSLRLSCVASGYSGYM
AWFRQAPGKEREGIAAIYRGDKITYYAHSVQGRFTISQANAKNTVYLLMNSLKPEDTAI
YYCAARRVVADSPLLSKTYAYWGQGTQVTVSSGSGKFERQVKKDQKDRVQG
2. SEQ ID NO: 2 - a-Synuclein Nb (Reference: PMID: 20620148)
MSGGSENLYFQCGGYPYDVPDYASGGQGQLVESGGGSVQAGGSLRLSCAASGIDSSSY
CMGWFRQRPGKEREGVARINGLGGVKTAYADSVKDRFTISRDNAENTVYLQMNSLKP
EDTAIYYCAAKFSPGYCGGSWSNFGYWGQGTQVTVSSGSGKFERQVKKDQKDRVQG
3. SEQ ID NO: 3 - a-Synuclein Nb PFF (Reference: PMID: 35853942)
MSGGSENLYFQCGGYPYDVPDYASGGQVQLQESGGGLVQAGGSLRLSLSASRYIFTLM
GMRWYRRAPGKERELVASIQVGSDTNYRDSVKGRFTLSRDNAKNTVYLQMNSLKSDD
TAVYYAAAPAYARRLHRYRGQGTQVTVSSGSGKFERQVKKDQKDRVQG
4. SEQ ID NO: 4 - TEV protease cleavage site
ENLYFQC
5. SEQ ID NO: 5 - TEV protease cleavage site
ENLYFQG
6. SEQ ID NO: 6 - Hemagglutinin (HA) tag
YPYDVPDYA
7. SEQ ID NO: 7 - a-Synuclein Nb
GQVQLQESGGGSVQTGGSLRLSCVASGYSGYMAWFRQAPGKEREGIAAIYRGDKITYY
AHSVQGRFTISQANAKNTVYLLMNSLKPEDTAIYYCAARRVVADSPLLSKTYAYWGQG TQVTVSS
8. SEQ ID NO: 8 - a-Synuclein Nb
GQGQLVESGGGSVQAGGSLRLSCAASGIDSSSYCMGWFRQRPGKEREGVARINGLGGV
KTAYADSVKDRFTISRDNAENTVYLQMNSLKPEDTAIYYCAAKFSPGYCGGSWSNFGY WGQGTQVTVSS 9. SEQ ID NO: 9 - chaperone-mediated autophagy 1 (CMA1) peptide motif KFERQVKKDQKDRVQ
10. SEQ ID NO: 10 - TDP-43 VH domain sequence (Reference: PMID: 29662239)
MSGGSENLYFQCGGYPYDVPDYASGGMEVQLQQSGAELVKPGASVKLSCTASGFNIKD YYMHWVKQRTEQGLEWIGRIDPEDGETKYAPKFQGKATITADTSSNTAYLQLSSLTSED TAVYYCTIIYYYGSRYVDYWGQGTTLTVSSGSGKFERQVKKDQKDRVQG
11. SEQ ID NO: 11 - TDP-43 VL domain sequence (Reference: PMID: 29662239) MSGGSENLYFQCGGYPYDVPDYASGGMEIVLTQSPTTMAASPGEKITITCSASSSISSSYL HWYQQKPGF SPKLLIYRTSNL ASGVPARF SGSGSGTS YSLTIGTMEAED VAT YYCQQGS SIPLTFGSGTKLEISSGSGKFERQVKKDQKDRVQG
12. SEQ ID NO: 12 - PelB-TDP-43VL domain sequence
MKYLLPTAAAGLLLLAAQPAMAGSMSGGSENLYFQCGGYPYDVPDYASGGMEIVLTQ SPTTMAASPGEKITITCS AS S SIS S SYLHWYQQKPGF SPKLLIYRTSNL ASGVPARF SGSGS GTSYSLTIGTMEAEDVATYYCQQGSSIPLTFGSGTKLEISSGSGKFERQVKKDQKDRVQ GLEVLFQGPSLEHHHHHH
13. SEQ ID NO: 13 - PelB-TDP-43VH-TEV-VL fusion sequence
MKYLLPTAAAGLLLLAAQPAMAGSMSGGSGYPYDVPDYASGGMEVQLQQSGAELVK
PGASVKLSCTASGFNIKDYYMHWVKQRTEQGLEWIGRIDPEDGETKYAPKFQGKATIT ADTSSNTAYLQLSSLTSEDTAVYYCTIIYYYGSRYVDYWGQGTTLTVSSGSGSGGGGEN LYFQGMEIVLTQSPTTMAASPGEKITITCSASSSISSSYLHWYQQKPGFSPKLLIYRTSNL ASGVPARF SGSGSGTS YSLTIGTMEAED VAT YYCQQGS SIPLTFGSGTKLEISSGSGKFER
QVKKDQKDRVQGLEVLFQGPSLEHHHHHH
14. SEQ ID NO: 14 - PelB leader sequence
KYLLPTAAAGLLLLAAQPAMA
15. SEQ ID NO: 15 - 3C protease cleavage site LEVLFQGP 16. SEQ ID NO: 16 - 6X Histidine tag
HHHHHH
17. SEQ ID NO: 17 - TDP-43VH domain
MEVQLQQSGAELVKPGASVKLSCTASGFNIKDYYMHWVKQRTEQGLEWIGRIDPEDGE TKYAPKFQGKATITADTSSNTAYLQLSSLTSEDTAVYYCTIIYYYGSRYVDYWGQGTTL TVSS
18. SEQ ID NO: 18 - TDP-43VL domain
MEIVLTQ SPTTM AASPGEKITITC S AS S SIS S S YLHW YQQKPGF SPKLLIYRTSNL ASGVP A RFSGSGSGTSYSLTIGTMEAEDVATYYCQQGSSIPLTFGSGTKLEISS
19. SEQ ID NO: 19 - HER2-Nb 2rsl5d (Reference: PMID: 21478264)
MQVQLQESGGGSVQAGGSLKLTCAASGYIFNSCGMGWYRQSPGRERELVSRISGDGDT WHKESVKGRFTISQDNVKKTLYLQMNSLKPEDTAVYFCAVCYNLETYWGQGTQVTVS S
20. SEQ ID NO: 20 - HER2-Nb 5F7 (Reference: PMID: 23159171)
MEVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTY YADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCKRFRTAAQGTDYWGQGTQVT vss
21. SEQ ID NO : 21 - HER2-Nb VHH 1028 (Reference : PMID : 35194100)
MEVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTY YADSVRGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCKRFRTAAQGTDYWGQGTQVT VSS
22. SEQ ID NO: 22 - HER2-Nb VHH17 (Reference: PMID: 34908139)
MQVQLVESGGGSVQAGGSLRLSCAASGYTYSSSCMGWFRQAPGKEREGVAGIKSTGG SKNYADSVKGRFTISQDNAKNTVYLQMNSLKPEDTAIYYCAAKYGGICSWGAILGVRE YNYWGQGTL VTVS S
23. SEQ ID NO: 23 - CMTM6 Nb 1A5 (Reference: PMID: 36418428) MQVQLQESGGGSVQAGGSLRLSCAASGYTYSNYCMGWFRQAPGKEREGVATIDRDGS
TNYAESVKGRFTISKDNAKNTLYLQMNSLKPEDTAMYYCAAVTWPGTCTVLAATSFG YWGQGTQ VTVSS
24. SEQ ID NO: 24 - HIF-la VHH212 (Reference: PMID: 33830713)
MVQLQESGGGSVQAGGSLRLSCVASGDTASMYCMGWFRQAPGKEREEVATIDSDGSV SIADSLKGRFTISKDSANNALYLHMNSLRPEDTANYYCAAGRPPCGSIFKPGYYYYGMD YWGKGTL VTVSS
25. SEQ ID NO: 25 - c-MYC VH-12-231 (Reference: PMID: 26319394)
MAEVQLLESGGGLVQPGGSLRLSCAASGFTFSRWGMSWVRQAPGKGLEWVSYISHDG TFIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGIIPRDLVGRLLLFDY WGQGTLVTVSS
26. SEQ ID NO: 26 - EGFR Nb 721D (Reference: PMID: 21520037)
MQVKLEESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDS TGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYW GQGTQVTVSS
27. SEQ ID NO: 27 - EGFR Nb 9G8 (Reference: PMID: 21520037)
MEVQLVESGGGL VQ AGGSLRLSC AASGRTF S S YAMGWFRQAPGKEREF VVAINWS SGS TYYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCAAGYQINSGNYNFKDYEY DYWGQGTQ VTVSS
28. SEQ ID NO: 28 - PCNA
MAQVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSDISPSGA
VKAYSDSVKGRFTISRDNAKNRLYLQMNSLTPEDTGEYFCTKVQSPRTRIPAPSSQGTQ VTVSS
29. SEQ ID NO: 29 - Linker peptide 1 and 2
CGGGSKGGGSKGGGS
30. SEQ ID NO: 30 - Linker peptide 3 and 4
GGGSKGGGSKGGGS 31. SEQ ID NO: 31 - Peptide carrier segment
GGGKG
32. SEQ ID NO: 32 - DBCO peptide
(PEG)nGnKGn wherein PEG refers to polyethylene glycol and n refers to number of PEG or G.
33. SEQ ID NO: 33 - chaperone-mediated autophagy 2 (CMA2) peptide motif KFERQKILDQRFFE
34. SEQ ID NO: 34 - Cell-penetrating peptide (CPP)
CI<RI<I<RRQRRRG
35. SEQ ID NO: 35 - Cell-penetrating peptide (CPP) rRrGrKkRr wherein r refers to D-Arginine and k refers to D-lysine.
36. SEQ ID NO: 36 - Cell-penetrating peptide (CPP)
RrRrRrRrRr wherein r refers to D-Arginine.

Claims

CLAIMS What is claimed is:
1. A nanodegrader (ND) composition comprising a chimeric nanobody (Nb) and a pharmaceutically acceptable carrier, wherein the Nb is fused to a cell penetrating peptide (CPP), a targeting peptide, and a proximity-enabled crosslinker (PEC).
2. The ND of claim 1, wherein the Nb comprises less than 200 amino acids or is less than 15kDa.
3. The ND of claim 1 or 2, wherein the Nb does not comprise a lysine residue.
4. The ND of any one of claims 1-3, wherein the Nb comprises at least one protease-resistant D-amino acid.
5. The ND of any one of claims 1-4, wherein the Nb comprises SEQ ID NO: 1, SEQ ID NO:
2, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO:
12, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, or a variant thereof.
6. The ND of any one of claims 1-5, wherein the CPP comprises a cyclic CPP or a membrane translocation domain (MTD).
7. The ND of any one of claims 1-6, wherein the CPP comprises an arginine-rich cyclic CPP.
8. The ND of any one of claims 1-7, wherein the CPP comprises CPP12, or a variant thereof.
9. The ND of any one of claims 1-8, wherein the CPP comprises SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, or a variant thereof.
10. The ND of any one of claims 1-9, wherein the targeting peptide comprises a chaperon- mediated autophagy (CMA) peptide, a macrocyclic (MC) peptide, E3 ligase ligand, or derivatives thereof.
11. The ND of any one of claims 1-10, wherein the PEC comprises a proximity-enabled sulfurfluoride exchange (SuFEx) crosslinker.
12. The ND of any one of claims 1-11, wherein the Nb is irreversibly bound to a protein of interest (POI) by the PEC.
13. The ND of claim 1-12, wherein the POI comprises a misfolded protein selected from tau, TAR DNA-binding protein 43 (TDP-43), a-synucleic, P-amyloid, prion proteins, human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR), CKLF like MARVEL transmembrane domain-containing 6 (CMTM6), human transcription factor cellular MYC (c-MYC), proliferating cell nuclear antigen (PCNA), or hypoxia-inducible factor 1 -alpha (HIF-la).
14. The ND of any one of claims 1-13, wherein the pharmaceutically acceptable carrier comprises an excipient, a diluent, a salt, a buffer, a stabilizer, a lipid, an emulsion, or a native Nb.
15. The ND of any one of claims 1-14, further comprising one or more protein labeling compounds.
16. The ND of claim 15, wherein the protein labeling compound comprises biotin, a reporter enzyme, a fluorophore, or a radioactive isotope.
17. A vector comprising a nucleic acid sequence encoding the ND, or a fragment thereof, of any one of claims 1-16.
18. A kit comprising the ND, the vector, or a salt thereof, of any one of claims 1-17.
19. The kit of claim 18, further comprising one or more reagents for stabilizing the ND.
20. The kit of claim 18 or 19, further comprising one or more reagents for delivering the ND to a biological sample or subject.
21. The kit of any one of claims 18-20, further comprising one or more reagents for amplifying the vector.
22. The kit of any one of claims 18-21, further comprising one or more reagents for expressing the vector in a cell.
23. A method of treating or preventing a disease in a subject in need thereof, the method comprises administering a pharmaceutically effective amount of a nanodegrader (ND) composition comprising a chimeric nanobody (Nb) and a pharmaceutically acceptable carrier, wherein the Nb is operably linked to a cell penetrating peptide (CPP), a targeting peptide, a proximity-enabled crosslinker (PEC), and wherein the ND targets and degrades a disease-related protein.
24. The method of claim 23, wherein the Nb comprises less than 200 amino acids or is less than 15kDa.
25. The method of clam 23 or 24, wherein the Nb does not comprise a lysine residue.
26. The method of any one of claims 23-25, wherein the Nb comprises at least one proteaseresistant D-amino acid.
27. The method of any one of claims 23-26, wherein the Nb comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, or a variant thereof.
28. The method of any one of claims 23-27, wherein the CPP comprises a cyclic CPP or a membrane translocation domain (MTD).
29. The method of any one of claims 23-28, wherein the CPP comprises an arginine-rich cyclic CPP.
30. The method of any one of claims 23-29, wherein the CPP comprises CPP12, or a variant thereof.
31. The method of any one of claims 23-30, wherein the CPP comprises SEQ ID NO: 34, SEQ
ID NO: 35, SEQ ID NO: 36, or a variant thereof.
32. The method of any one of claims 23-31, wherein the targeting peptide comprises a chaperon-mediated autophagy (CMA) peptide, a macrocyclic (MC) peptide, E3 ligase ligand, or derivatives thereof.
33. The method of any one of claims 23-32, wherein the PEC comprises a proximity-enabled sulfur-fluoride exchange (SuFEx) crosslinker.
34. The method of any one of claims 23-33, wherein the Nb is irreversibly bound to the disease-related protein by the PEC.
35. The method of any one of claims 23-34, wherein the disease-related protein comprises a misfolded protein or a cancer-associated protein selected from tau, TAR DNA-binding protein 43 (TDP-43), a-synucleic, P-amyloid, a prion protein, human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR), CKLF like MARVEL transmembrane domain-containing 6 (CMTM6), human transcription factor cellular MYC (c-MYC), proliferating cell nuclear antigen (PCNA), or hypoxia-inducible factor 1-alpha (HIF-la).
36. The method of any one of claims 23-35, wherein the pharmaceutically acceptable carrier comprises an excipient, a diluent, a salt, a buffer, a stabilizer, a lipid, an emulsion, or a native Nb.
37. The method of any one of claims 23-36, further comprising one or more protein labeling compounds.
38. The method of claim 37, wherein the protein labeling compound comprises biotin, a reporter enzyme, a fluorophore, or a radioactive isotope.
39. The method of any one of claims 23-38, wherein the method decreases or reduces the disease-related protein relative to an untreated control.
40. The method of any one of claims 23-39, further comprising administering the ND and a therapeutic agent.
41. The method of claim 40, wherein the therapeutic agent comprises an antibiotic, an antiinflammatory compound, a sedative, an anesthetic, an anti-viral agent, a peptide hormone, an antidiabetic agent, a steroid, or combinations thereof.
42. The method of any one of claims 23-41, wherein the disease comprises a neurodegenerative disease or a cancer.
43. The method of any one of claims 23-42, wherein the neurodegenerative disease comprises Alzheimer’s disease, ataxia, Huntington’s disease, Parkinson’s disease, amyotrophic lateral sclerosis (ALS), Friedreich ataxia, Lewy body disease, a prion disease, or Creutzfeldt-Jakob disease.
44. The method of any one of claims 23-43, wherein the cancer comprises lung cancer, breast cancer, gastric cancer, brain cancer, gliomas, or neuroblastoma.
45. A method of degrading or eliminating a disease-related protein in a subject or biological sample, the method comprising: a. introducing a nanodegrader (ND) composition to the subject or biological sample, wherein the ND comprises a chimeric nanobody (Nb) and pharmaceutically acceptable carrier, wherein the Nb is operably linked to a cell penetrating peptide (CPP), a targeting peptide, a proximity-enabled crosslinker (PEC); b. internalizing the ND into a cell of the subject or biological sample, wherein the CPP directs internalization into the cell; c. irreversibly targeting and binding the ND to the disease-related protein, wherein the PEC binds the disease-related protein to the ND; and d. degrading the disease-related protein, wherein the targeting peptide activates at least one proteolytic pathway to decrease the disease-related protein relative to a control subject or biological sample.
46. The method of claim 45, wherein the Nb comprises less than 200 amino acids or is less than 15kDa.
47. The method of clam 45 or 46, wherein the Nb does not comprise a lysine residue.
48. The method of any one of claims 45-47, wherein the Nb comprises at least one proteaseresistant D-amino acid.
49. The method of any one of claims 45-48, wherein the Nb comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, or a variant thereof.
50. The method of any one of claims 45-49, wherein the CPP comprises a cyclic CPP or a membrane translocation domain (MTD).
51. The method of any one of claims 45-50, wherein the CPP comprises an arginine-rich cyclic CPP.
52. The method of any one of claims 45-51, wherein the CPP comprises CPP12, or a variant thereof.
53. The method of any one of claims 45-52, wherein the CPP comprises SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, or a variant thereof.
54. The method of any one of claims 45-53, wherein the targeting peptide comprises a chaperon-meduated autophagy (CMA) peptide, a macrocyclic (MC) peptide, E3 ligase ligand, or derivatives thereof.
55. The method of any one of claims 45-54, wherein the PEC comprises a proximity-enabled sulfur-fluoride exchange (SuFEx) crosslinker.
56. The method of any one of claims 45-55, wherein the Nb is irreversibly bound to the disease-related protein by the PEC.
57. The method of claim 56, wherein the disease-related protein comprises a misfolded protein or a cancer-associated protein selected from tau, TAR DNA-binding protein 43 (TDP-43), a- synucleic, P-amyloid, prion proteins, human epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor (EGFR), CKLF like MARVEL transmembrane domaincontaining 6 (CMTM6), human transcription factor cellular MYC (c-MYC), proliferating cell nuclear antigen (PCNA), or hypoxia-inducible factor 1-alpha (HIF-la).
58. The method of any one of claims 45-57, wherein the pharmaceutically acceptable carrier comprises an excipient, a diluent, a salt, a buffer, a stabilizer, a lipid, an emulsion, or a native Nb.
59. The method of any one of claims 45-58, further comprising one or more protein labeling compounds.
60. The method of claim 59, wherein the protein labeling compound comprises biotin, a reporter enzyme, a fluorophore, or a radioactive isotope.
61. The method of any one of claims 45-60, wherein the at least one proteolytic pathway comprises a ubiquitin-proteosome pathway, a lysosomal proteolytic pathway, or a combination thereof.
62. The method of any one of claims 45-61, wherein the method treats or prevents a disease selected from Alzheimer’s disease, ataxia, Huntington’s disease, Parkinson’s disease, amyotrophic lateral sclerosis (ALS), Friedreich ataxia, Lewy body disease, a prion disease, Creutzfeldt-Jakob disease, lung cancer, breast cancer, gastric cancer, brain cancer, gliomas, or neuroblastoma.
63. The method of any one of claims 45-62, wherein the biological sample comprises a tissue biopsy, a cerebrospinal fluid (CSF) sample, a blood sample, a serum sample, or an isolated cell.
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