EP4774005A1 - Compositions for transport of therapeutic cargos using miniprotein binders targeting ca-iv - Google Patents

Compositions for transport of therapeutic cargos using miniprotein binders targeting ca-iv

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Publication number
EP4774005A1
EP4774005A1 EP24863681.3A EP24863681A EP4774005A1 EP 4774005 A1 EP4774005 A1 EP 4774005A1 EP 24863681 A EP24863681 A EP 24863681A EP 4774005 A1 EP4774005 A1 EP 4774005A1
Authority
EP
European Patent Office
Prior art keywords
seq
sequence
miniprotein
conjugate
receptor
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.)
Pending
Application number
EP24863681.3A
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German (de)
French (fr)
Inventor
Viviana Gradinaru
Xiaozhe DING
Seongmin Jang
Xinhong Chen
Erin E. SULLIVAN
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California Institute of Technology
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California Institute of Technology
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Publication of EP4774005A1 publication Critical patent/EP4774005A1/en
Pending legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • compositions for Transport of Therapeutic Cargos Using Miniprotein Binders Targeting CA-IV Compositions for Transport of Therapeutic Cargos Using Miniprotein Binders Targeting CA-IV
  • the invention relates to methods and shuttles for crossing the blood brain barrier.
  • the blood brain barrier presents a fundamental bottleneck to the development of effective research tools and therapeutics for the central nervous system (CNS).
  • This structure comprising mainly of brain endothelial cells, requires large molecules to be delivered via invasive intracranial injections, technically challenging focused ultrasound, or receptor-mediated transcytosis.
  • the rational design of BBB-crossing large molecules has long been hampered by the imperfect understanding of the mechanisms involved in transcytosis, with only a handful of targets, such as the transferrin receptor, validated for research and therapies.
  • compositions and methods comprising miniproteins as shuttles inducing transcytosis across target tissues, for example through an astrocyte-specific receptor, neuron-specific receptor, carbonic anhydrase IV (CA-IV), low density lipoprotein receptor related protein 6 (LRP6), or lymphocyte antigen 6 complex, locus A (LY6A, also known as stem cell antigen- 1 (SCA-1)).
  • miniproteins as shuttles inducing transcytosis across target tissues, for example through an astrocyte-specific receptor, neuron-specific receptor, carbonic anhydrase IV (CA-IV), low density lipoprotein receptor related protein 6 (LRP6), or lymphocyte antigen 6 complex, locus A (LY6A, also known as stem cell antigen- 1 (SCA-1)).
  • the present invention provides miniproteins, typically between 5-20 kD in size, that can be modularly fused to various therapeutic cargos without disrupting functions of the cargos.
  • Their epitope-informed design of miniproteins of the invention ensures they bind effectively to relevant sites. Coupled with a strengthened affinity through an expanded binding interface and favorable drug-like properties such as thermostability and high solubility, miniproteins represent a promising avenue for targeted therapeutic delivery. Further advantageously, miniproteins of the invention can be cost-effectively produced in E. coli.
  • aspects of the invention provide a conjugate comprising a miniprotein shuttle which binds to a portion of a transcytosis-mediating cell receptor and a therapeutic cargo conjugated to the shuttle.
  • the shuttle mediates transcytosis across the receptor, for example an astrocyte-specific receptor, neuron-specific receptor, CA-IV, LRP6, or LY6A.
  • nucleic acid molecules and peptides may comprise one or more substitutions, for example conservative substitutions, that allow sequences to continue to function. Accordingly, sequences may have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence disclosed.
  • a conservative substitution refers to amino acid substitutions that do not significantly affect or alter binding characteristics of a particular protein.
  • conservative substitutions are ones in which a substituted amino acid residue is replaced with an amino acid residue having a similar side chain.
  • conservative substitutions may include a substitution found in one of the following groups: Group 1 : Alanine (Ala or A), Glycine (Gly or G), Serine (Ser or S), Threonine (Thr or T); Group 2: Aspartic acid (Asp or D), Glutamic acid (Glu or Z); Group 3: Asparagine (Asn or N), Glutamine (Gin or Q); Group 4: Arginine (Arg or R), Lysine (Lys or K), Histidine (His or H); Group 5: Isoleucine (He or I), Leucine (Leu or L), Methionine (Met or M), Valine (Vai or V); and Group 6: Phenylalanine (Phe or F), Tyrosine (Tyr or
  • amino acids can be grouped into conservative substitution groups by similar function, chemical structure, or composition (e.g., acidic, basic, aliphatic, aromatic, or sulfur-containing).
  • an aliphatic grouping may include, for purposes of substitution, Gly, Ala, Vai, Leu, and He.
  • Other conservative substitutions groups include sulfur- containing: Met and Cysteine (Cys or C); acidic: Asp, Glu, Asn, and Gin; small aliphatic, nonpolar, or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; polar, negatively charged residues and their amides: Asp, Asn, Glu, and Gin; polar, positively charged residues: His, Arg, and Lys; large aliphatic, nonpolar residues: Met, Leu, He, Vai, and Cys; and large aromatic residues: Phe, Tyr, and Trp.
  • Met and Cysteine Cys or C
  • acidic Asp, Glu, Asn, and Gin
  • small aliphatic, nonpolar, or slightly polar residues Ala, Ser, Thr, Pro, and Gly
  • polar, negatively charged residues and their amides Asp, Asn, Glu, and Gin
  • polar, positively charged residues His, Arg, and Lys
  • the miniprotein may bind the transcytosis-mediating cell receptor comprises a sequence having at least 95% sequence with a sequence selected from SEQ ID NO: 1-248.
  • the miniprotein may bind the transcytosis-mediating cell receptor LY6A and comprises a sequence having at least 95% sequence with a sequence selected from SEQ ID NO: 1-16, SEQ ID NO: 29-31, SEQ ID NO: 41, SEQ ID NO: 52, SEQ ID NO: 122, SEQ ID NO: 128-129, SEQ ID NO: 139-142, or SEQ ID NO: 144-147.
  • the miniprotein may bind the transcytosis-mediating cell receptor CA-IV and comprises a sequence having at least 95% sequence with a sequence selected from SEQ ID NO: 17-28, SEQ ID NO: 32-37, SEQ ID NO: 42-49, SEQ ID NO: 54-92, SEQ ID NO: 110-121, SEQ ID NO: 123-125, SEQ ID NO: 131-138, SEQ ID NO: 143, SEQ ID NO: 148, or SEQ ID NO: 171- 248.
  • the miniprotein may bind the transcytosis-mediating cell receptor LRP6 and comprises a sequence having at least 95% sequence with a sequence selected from SEQ ID NO: 38-40, SEQ ID NO: 44, SEQ ID NO: 53, SEQ ID NO: 93-109, SEQ ID NO: 126, SEQ ID NO: 130, SEQ ID NO: 149-170, or SEQ ID NO: 225-227.
  • the miniprotein may bind an astrocyte-specific or neuron-specific receptor and comprises a sequence having at least 95% sequence with a sequence selected from SEQ ID NO: 50-51 or SEQ ID NO 127.
  • the therapeutic cargo may be conjugated to the shuttle via a linker.
  • Therapeutic cargo may covalently conjugated to the shuttle.
  • the therapeutic cargo may be a biological molecule, for example a nucleic acid (for example, RNA, siRNA, DNA, or an ASO), a protein (for example, an enzyme), a peptide, an antibody, a nanobody, a lipid, a polysaccharide, and a combination thereof.
  • the therapeutic cargo may be a non-biological molecule, for example a small molecule or a dye.
  • the payload may comprise DNA molecules, oligonucleotides, therapeutic proteins, small molecule therapeutics, interfering RNA, gene editing cargo, chemotherapeutic agents, toxins, radioisotopes, enzymes, chelators, boron compounds, photoactive agents, dyes, metals, metal alloys, nanoparticles, or other larger synthetic molecules and biologies in vitro and in vivo.
  • the miniprotein may be in any known form, for example as described hereinbelow.
  • the miniprotein may be in a monomeric form, bi-specific form , multi-specific form, or as part of an alternative protein scaffolds.
  • the miniprotein may be cystine-reinforced, hydrophobic core, or chemically-stabilized
  • the conjugate may be characterized by delivery of the therapeutic cargo across the blood brain barrier (BBB).
  • BBB blood brain barrier
  • the therapeutic cargo may be therapeutic cargo for the treatment of a disorder affecting the central nervous system.
  • the conjugate may facilitate transcytosis across a target tissue including brain or eye tissue.
  • miniproteins may also be provided with or without other protein scaffolds in other delivery systems, including viral vectors (c. ., lentivirus, adenovirus, AAVs), non-viral nanoparticles, exosomes, antibodies, antibody-drug conjugates, or proteins.
  • viral vectors c. ., lentivirus, adenovirus, AAVs
  • non-viral nanoparticles e. ., lentivirus, adenovirus, AAVs
  • exosomes e. lentivirus, adenovirus, AAVs
  • aspects of the invention further provide methods and uses that comprise delivery of a therapeutic cargo across a transcytosis-mediating receptor of a subject.
  • Methods and uses of the invention (including in the formulation of a medicament), comprise providing to a subject a conjugate of the invention, as described throughout this application, comprising a miniprotein shuttle of the invention and a therapeutic cargo conjugated to the shuttle.
  • FIG. 1 is a workflow for the design and screen of miniproteins of the invention.
  • FIG. 2A-B are images from Western blots from the synthesis of miniproteins of the invention.
  • FIG. 3A-H are graphs of surface plasmon resonance (SPR) curves of LY6A-binding miniproteins of the invention.
  • FIG. 4AA-4BT are graphs of SPR curves of CA-IV-binding miniproteins of the invention.
  • FIG. 5A-5J are graphs of SPR curves of LRP6-binding miniproteins of the invention.
  • FIG. 6A-D are images of fluorescent imaging of in vitro internalization of LY6A-binding miniproteins of the invention.
  • FIG. 7A-B are images of fluorescent imaging of in vitro internalization of CA-IV- binding miniproteins of the invention.
  • FIG. 8A-B are images of fluorescent imaging of in vitro internalization of LY6A-binding miniproteins of the invention.
  • FIG. 9A-F are graphs of SPR curves of purified miniproteins binders at different concentrations.
  • FIG. 10A-E show sagittal slices of tissues from mice injected with miniproteins of the invention.
  • compositions and methods comprising miniproteins as shuttles inducing transcytosis across target tissues, for example through an astrocyte-specific receptor, neuron-specific receptor, carbonic anhydrase IV (CA-IV), low density lipoprotein receptor related protein 6 (LRP6), or lymphocyte antigen 6 complex, locus A (LY6A).
  • miniproteins as shuttles inducing transcytosis across target tissues, for example through an astrocyte-specific receptor, neuron-specific receptor, carbonic anhydrase IV (CA-IV), low density lipoprotein receptor related protein 6 (LRP6), or lymphocyte antigen 6 complex, locus A (LY6A).
  • Miniproteins “computationally designed binders,” “small-sized binders,” used interchangeably herein, are a diverse group of protein scaffolds characterized by small size, stability, and versatility in drug-like roles.
  • miniproteins may contain stable tertiary structures that facilitate specificity and potency in addition to resistance to proteolysis, reduction, and denaturation while also being of sufficiently small size that synthetic production, complete characterization, and regulation as a small molecule drug rather than as a biological product is possible. Their size also facilitates computational design and SAR as well as favorable penetration through tissue and manufacturing flexibility.
  • miniproteins can be segregated into three general subcategories, based on biophysical properties that impact their pharmacologic capabilities and biosynthetic production strategies: cystine-reinforced, hydrophobic core, and chemically-stabilized
  • the largest miniproteins are the hydrophobic core miniproteins, whose structure and stability are driven by rigid secondary structural elements (a-helix and 0-sheet) arranged around a hydrophobic core whose solvent exclusion drives its folding.
  • hydrophobic core miniproteins examples include affibodies, adnectin, centyrins, nanofittins, affitins, and funomers.
  • Adnectins and centyrins are based on fibronectin type 3 (10Fn3) domains, with similarities to antibody VH domains with prominent loops extending out from a quasi-P-barrel structure.
  • Fynomers are adapted from the SH3 domain of Fyn kinase and are primarily P-barrels with exposed loops for binding.
  • Affibodies are simple three-helix domains based on the antibody-engaging Z-domain of Protein A.
  • Nanofittins and affitins are adapted from various 7- kDa DNA binding proteins from thermophilic archea and possess extraordinary thermal stability. All four classes have no need for chemical or oxidative stapling; as such, they can be produced in bacteria in large quantities.
  • the binding of hydrophobic core miniproteins is mainly driven by similar strategies as in antibody Fv domains, comprising randomized sequences within loops or helices attached to a rigid superstructure.
  • Cystine-reinforced miniproteins use disulfides to drive folding and provide rigidity, with their folding and stability dependent on Cys-Cys disulfides.
  • cystine-reinformed miniproteins examples include avimers, kunitdomains, and cystine dense peptides (CDPs).
  • Cystine-dense peptides are represented by structurally diverse scaffolds that have similar biophysical properties, including native naturally cell-penetrant or blood-brain barrierpenetrant peptides.
  • the striking protease resistance provided by the cystine knot can enable activity in aggressive environments such as the gastrointestinal (GI) tract.
  • Kunitz domains have similarities to CDPs but contain a hydrophobic core and are specialized for protease inhibition; hundreds of such proteins exist in nature, such as the popular engraftment scaffold human APPI.
  • Avimers are based on the loop-rich A-domains of human cell surface receptors, and, unlike CDPs or Kunitz domains, require coordination of a calcium ion.
  • Avimers have been shown to be highly amenable to multimerization. While being smaller than hydrophobic core miniproteins, the screening strategies for cystine-reinforced miniproteins are similar to that of larger scaffolds. Binding is imparted by identifying surface exposed regions to engraft known binder motifs or randomized sequences, or by whole-protein (sparing cysteines) mutational screening.
  • Chemically stabilized miniproteins are the most similar to small molecule drugs in both size and functionality. Chemically stabilized miniproteins have unique properties but can roughly be considered as miniature, rigidified versions of 0-sheets (P-hairpins), a-helices (stapled peptides), or loops (bicycles) found at native protein-protein interfaces. All rely on a chemical crosslinking step to stabilize what would otherwise be unstable or unstructured peptide sequences.
  • chemically stabilized miniproteins include P-hairpins, stapled peptides, and bicycles.
  • a payload e.g., a therapeutic agent
  • a transcytosis-mediating receptor for example an astrocyte-specific receptor, neuron-specific receptor, CA-IV, LRP6, or LY6A.
  • the method comprises providing a miniprotein capable of interacting with an astrocytespecific receptor, neuron-specific receptor, CA-IV, LRP6, or LY6A.
  • the miniprotein can be part of a delivery system and the delivery system can comprise a payload to be delivered to a nervous system.
  • the method can further comprise administering the delivery system to the subject.
  • the delivery system comprises nanoparticles, nanotubes, nanowires, dendrimers, liposomes, ethosomes and aquasomes, polymersomes and niosomes, foams, hydrogels, cubosomes, quantum dots, exosomes, macrophages, and combinations thereof.
  • the delivery system comprises a nanoparticle selected from lipid-based nanoparticles, polymeric nanoparticles, inorganic nanoparticles, surfactant-based emulsions, nanowires, silica nanoparticles, virus-like particles, peptide or protein-based particles, lipidpolymer particles, nanolipoprotein particles, and combinations thereof.
  • the payload may include an antimicrobial agent, a therapeutic agent, a prodrug, a peptide, a protein, an enzyme, a lipid, a biological response modifier, a pharmaceutical agent, a lymphokine, a heterologous antibody or fragment thereof, a detectable label, a polyethylene glycol (PEG) molecule, or a combination of two or more of the agents.
  • an antimicrobial agent e.g., a therapeutic agent, a prodrug, a peptide, a protein, an enzyme, a lipid, a biological response modifier, a pharmaceutical agent, a lymphokine, a heterologous antibody or fragment thereof, a detectable label, a polyethylene glycol (PEG) molecule, or a combination of two or more of the agents.
  • PEG polyethylene glycol
  • the payload can include a neuroactive polypeptide, for example, a neurotrophic factors, endocrine factors, growth factors, paracrine factors, hypothalamic release factors, neurotransmitter polypeptides, polypeptide agonists for a receptor expressed by a CNS cell, polypeptides involved in lysosomal storage disease or any combination thereof.
  • a neuroactive polypeptide for example, a neurotrophic factors, endocrine factors, growth factors, paracrine factors, hypothalamic release factors, neurotransmitter polypeptides, polypeptide agonists for a receptor expressed by a CNS cell, polypeptides involved in lysosomal storage disease or any combination thereof.
  • the payload can include an IL-1 receptor antagonist (IL-IRa), dalargin, an interferon-P, Glial-derived neurotrophic factor (GDNF), tumor necrosis factor receptor (TNFR), nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin-4/5, neurotrophin (NT)-3, a neurturin, neuregulin, a netrin, ciliary neurotrophic factor (CNTF), stem cell factor (SCF), a semaphorin, hepatocyte growth factor (HGF), epidermal growth factor (EGF), transforming growth factor (TGF)-cx, TGF-B, vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), heregulin, artemin, persephin, interleukins, granulocyte-colony stimulating factor (CSF), granulocyte-macrophage-CSF, cardiotrophin-1, hedgehogs, leukemia inhibitory factor
  • aspects of the invention also provide for delivery of the conjugate to a subject in order to transport a therapeutic agent across the BBB.
  • delivery of the therapeutic payload may be for the treatment of a disease, disorder, or injury of the CNS.
  • the therapeutic agent may be released from the conjugate following entry into the CNS.
  • the disease, disorder, or injury of the CNS can be, without limitation, multiple sclerosis (MS), amylotrophic lateral sclerosis (ALS), Huntington's disease, Alzheimer's disease, Parkinson's disease, spinal cord injury, traumatic brain injury, stroke, neuropathic pain, neurodegeneration, neuroinflammation, progressive multifocal leukoencephalopathy (PML), encephalomyelitis (EPL), central pontine myelolysis (CPM), adrenoleukodystrophy, Alexander's disease, Pelizaeus Merzbacher disease (PMZ), Globoid cell Leucodystrophy (Krabbe's disease), Wallerian Degeneration, optic neuritis, transverse myelitis, post radiation injury, neurologic complications of chemotherapy, acute ischemic optic neuropathy, vitamin E deficiency, isolated vitamin E deficiency syndrome, Bassen-Kornzweig syndrome, Marchiafava-Bignami syndrome, metachromatic leukodystrophy,
  • MS
  • CAs carbonic anhydrases
  • the carbonic anhydrases are a family of enzymes that catalyze the interconversion between carbon dioxide and water and the dissociated ions of carbonic acid.
  • CAs participate in a variety of biological processes, including respiration, calcification, acid-base balance, bone resorption, and the formation of aqueous humor, cerebrospinal fluid, saliva, and gastric acid.
  • Carbonic anhydrase IV is an isozyme that belongs to the carbonic anhydrase family, a family of zinc metalloenzymes, which catalyzes the reversible reaction of hydration of CO2 (TBO+CCh ⁇ HCCh — I-H+), allowing the enzyme to regulate intra- and extra-cellular concentrations of CO2, H+, and HCO3 -.
  • the carbonic anhydrases participate in a variety of biological processes, including respiration, calcification, acid-base balance, bone resorption, and the formation of aqueous humor, cerebrospinal fluid, saliva, and gastric acid. The carbonic anhydrases show extensive diversity in tissue distribution and in their subcellular localization.
  • I- VII there are at least seven genetically distinct isozymes of mammalian carbonic anhydrase, designated I- VII, each of which catalyzes the reversible hydration of carbon dioxide by a zinchydroxide mechanism.
  • Physiological functions that are regulated by carbonic anhydrase comprise, for example, removal of HCO3- in lung by respiration, reutilization of HCO3- in kidney, production of aqueous humor in eyes, cerebrospinal fluids in brain, gastric juice production in stomach, pancreatic juice, and bone resorption by osteoclasts.
  • Carbonic anhydrase family members also play important roles in metabolic processes that include ureagenesis, gluconeogenesis, and lipogenesis.
  • carbonic anhydrase IV is a glycosylphosphatidylinositol-anchored membrane isozyme. Carbonic anhydrase IV is broadly conserved across vertebrates and has similar CNS expression profiles in humans, with a recent single cell analysis of human brain vasculature confirming CA-IV's expression in the human BBB. Carbonic anhydrase IV has been shown to regulate pH, which is associated with neural discharge and can influence neuronal function through ion-gated channels. In some embodiments, the carbonic anhydrase IV disclosed herein is a human carbonic anhydrase IV.
  • CA-IV is known to localize on the luminal surface of brain endothelial cells throughout the cortex and cerebellum where it enzymatically modulates carbon dioxidebicarbonate balance.
  • Human CA-IV has been previously characterized as a 35-kDa protein with a “high activity” in CO2 hydration and a higher activity than other isozymes in catalyzing the dehydration of HC03 -.
  • human CA-IV contains an 18-amino acid signal sequence at the N-terminal of the protein for endoplasmic reticulum (ER) translocation and a 260-amino acid “CA domain” containing active site amino acid residues that shows 30-36% homology with cytoplasmic CAs.
  • CA-IV contains no classical consensus sites (Asn-Xxx-Ser/Thr) for N-glycosylation.
  • Human CA-IV also contains no oligosaccharide chains, while other mammalian carbonic anhydrase IV (e.g. mouse carbonic anhydrases IV) are glycoproteins with one to several oligosaccharide side chains.
  • the carbonic anhydrase IV disclosed herein is a mouse carbonic anhydrase IV.
  • CA-IV has recently been found to be among the mouse proteins most strongly positively correlated with plasma-protein uptake in the brain (slightly stronger than the often- targeted transferrin receptor). This property is useful for identifying receptors for enhanced BBB crossing.
  • CA-IV is also expressed in the GI tract, kidney, and lung, as well as taste receptor cells where it allows the sensing of carbonation.
  • Mouse and human CA-IV are highly homologous, containing the same amino acids at positions crucial for enzyme activity (e.g., histidine residue 64 (His 64)), with several differences including, for example, that mouse CA-IV is an N-linked glycoprotein and the CO2 hydration rate catalyzed by mouse CA-IV is much lower than human CA-IV. Without being bound by any theory, the lower enzyme activity of mouse CA-IV may be associated with the replacement of Gly 63 in human CA-IV with Gin 63, among several other amino acid replacements.
  • Another difference between mouse and human CA-IV is the Val-131- Asp-136 segment (13O's segment) that forms an a-helix in mouse and an extended loop in human CA-IV.
  • a carbonic anhydrase IV disclosed herein as a receptor for enhancing BBB crossing can be any carbonic anhydrase IV, such as a mouse CA-IV, a human CA-IV, or a homology or a variant thereof.
  • Carbonic anhydrase IV homologs and/or variants can be derived from a vertebrate species including, but not limited to, mouse, rat, human, bovine, rabbit, monkey, pig, horse, rainbow trout, chimpanzee, squirrel, chicken, goat, and sheep.
  • Carbonic anhydrase IV homologs from various species can be found in public databases identifiable to a person skilled in the art, including for example UniProt, NCBI, and Swiss-Prot.
  • a miniprotein can interact with a carbonic anhydrase IV disclosed herein (e.g., mouse CA-IV, human CA-IV or a homology or a variant thereof), thereby increasing permeability of the BBB (e.g., through transcytosis).
  • a carbonic anhydrase IV disclosed herein e.g., mouse CA-IV, human CA-IV or a homology or a variant thereof
  • the increase in the permeability of the BBB is achieved by altering (e.g., increasing or decreasing) the carbonic anhydrase IV activity, such as reducing its activity.
  • the alteration of carbonic anhydrase IV activity is achieved by a shuttle interacting to one or more active sites of the carbonic anhydrase IV including the zinc binding site and the hydrophobic substrate binding pocket.
  • the shuttle can interacting with the zinc binding site, the hydrophobic substrate binding pocket, or both.
  • the zinc binding site in carbonic anhydrase IV has a conserved structure dominated by a P-sheet super-structure with a metal binding site formed by at least three His residues. Without being bound by any theory, it is believed that the zinc binding site is on one face of the P-sheet at the bottom of a 15-A-deep, conical active site cleft in which zinc is liganded by three His residues and hydroxide ion with tetrahedral geometry.
  • the hydrophobic substrate binding pocket is adjacent to zinc-bound hydroxide, formed in large part by bulky residues such as Vai at its base and Vai, Trp and Leu at its neck. This pocket is highly conserved among all active isozymes on the basis of phylogenetic comparisons.
  • the hydrophobic pocket has a minimum width and depth for efficient catalysis, and linear free energy relationships indicate that the volume of the amino acid residue at the base of the pocket and the hydrophobicity of residues at the neck of the pocket are critical for activity. Both the zinc binding site and the hydrophobic substrate binding pocket are highly conserved among carbonic anhydrase isozymes.
  • LRP6 low density lipoprotein receptor related protein 6
  • the LRP6 gene encodes a member of the low density lipoprotein receptor (LDLR) gene family, which consists of cell surface proteins involved in receptor-mediated endocytosis of specific ligands.
  • LDLR proteins are composed of the same basic structural motifs: an extracellular domain that contains LDLR binding repeats and EGF repeats with associated spacer domains containing the YWTD motif; a single transmembrane-spanning domain; and a C- terminal cytoplasmic domain that generally contains at least 1 copy of the NPXY motif.
  • Lrp6 is a single-pass transmembrane protein involved in the activation of Wnt signalling pathways.
  • the human LRP6 has 23 exons, is found in chromosome 12p 13.2, and contains 1613 amino acids.
  • LRP6 consists of four YWTD P-propeller domains, each of which is followed by an EGF-like domain, and this is followed by three LDLR type A repeats, a transmembrane domain, and a short intracellular domain. It is structurally related to LRP5, and they share almost 71% homology at the nucleotide level. In Lrp6, approximately 85% of the 1613-amino-acid length is extracellular. Most extracellular ligands bind LRP6 at the P-propellers. Each protein has a singlepass, 22-amino-acid segment that crosses the cell membrane and a 207-amino-acid segment that is internal to the cell. lymphocyte antigen 6 complex, locus A (LY6A)
  • Sca-1 is among the first identified members of the murine Ly6 gene family.
  • the Ly6 gene family belongs to the superfamily of lymphocyte antigen-6 (Ly6)/urokinase-type plasminogen activator receptor (uPAR) proteins. This superfamily is characterized by the presence of LU domain.
  • LU domain is a 60-80 amino acid domain, which is composed of 6-10 cysteines arranged in a specific spacing pattern that allows distinct disulfide bridges which create the three-fingered (3F) structural motif.
  • human chromosome 8 the syntenic region to mouse chromosome 15 — harbors several genes containing the characteristic s LU domain.
  • the LU domain is found in extracellular domains of cell-surface receptors with membrane spanning domain (activin type 2 receptor and bone morphogenetic type IA receptor), or in GPI-anchored proteins CD 177 or in secreted globular proteins such CD59 antigen, SLURP 1/2.
  • Miniprotein binders were computational designed and tailored for specific receptors not only for crossing the blood brain barrier (BBB) but also for receptor-mediated delivery to other organs.
  • BBB blood brain barrier
  • the proprietary design methodology behind these miniproteins ensured they were epitope-informed, offering precise binding to targeted receptors.
  • These designed miniproteins may enable specific and efficient therapeutic agent delivery.
  • miniprotein binders were designed targeting different BBB receptors, such as mouse LY6A, human CA-IV, and human LRP6 and screened against their targets. It was shown using surface plasmon resonance (SPR) that miniproteins of the invention bind to their target receptors. Miniproteins were shown to further be internalized in cells overexpressing the target receptor. To demonstrate the in vivo application potential of the miniproteins binders, the in vivo distribution of a miniprotein targeting a BBB receptor found in rodent brains was characterized.
  • SPR surface plasmon resonance
  • FIG. 1 is a three-stage workflow for the design and screen of miniproteins of the invention.
  • stage 1 the receptor target was analyzed to formulate a design strategy: la) Binding motif identification: experimentally validated receptor binding motifs were identified by either literature or an in vitro pull-down selection; lb) Binding structure analysis: The complex structure of the binding motif and the receptor was obtained using either the protein data bank (PDB) database or predictive tools like AlphaFol d-Multimer; lc) Design strategy formation: A design strategy, such as epitope targeting or motif scaffolding, was determined based on available information.
  • PDB protein data bank
  • Backbone generation Backbone structure models were generated using a diffusionbased generative Al, for example Rfdiffusion;
  • stage 3 the designed sequences were screened and characterized:
  • the produced protein was screened using surface plasmon resonance (SPR) or a cellbased binding assay;
  • FIG. 2A-B are images from Western blots from the synthesis of miniproteins of the invention.
  • the blots were stained with 1 : 1000 anti-HA-HRP and bands were developed using UltraTMB substrate. Results indicated that the majority of designed miniproteins can be successfully produced to a concentration of 0.5-5 micromolar.
  • FIG. 3A-P are graphs of SPR curves of LY6A-binding miniproteins of the invention.
  • the designed miniproteins particularly the circled miniproteins (XMP-6, XMP-7, XMP- 34), showed binding signals. Note that the y-axis range and sample concentrations may differ between panels. 200 nM of Fc-tagged human CA-IV receptor proteins were diluted in HBS-EF+ buffer with 1% BSA were immobilized on a Protein A chip. Miniproteins synthesized with the cell-free reactions, diluted in the same buffer as receptor proteins, were then flown to the chip.
  • FIG. 4AA-4BT are graphs of SPR curves of CA-IV-binding miniproteins of the invention.
  • the designed miniproteins particularly the circled miniproteins (XMP-27 and XMP-52), showed binding signals. Note that the y-axis range and sample concentrations may differ between panels.
  • FIG. 5A-5J are graphs of SPR curves of LRP6-binding miniproteins of the invention.
  • the designed miniproteins particularly the circled miniproteins (XMP-42 and XMP-41), showed binding signals. Note that the y-axis range and sample concentrations may differ between panels.
  • FIG. 6A-D are images of fluorescent imaging of in vitro internalization of LY6A-binding miniproteins of the invention.
  • images of HEK293 cells incubated with miniproteins XMP-6 showed specific enrichment of XMP-6 in cells overexpressing LY6A receptor.
  • a Z-slice of representative cells showed internal granular signals that likely indicate endocytosis.
  • images of HEK293 cells incubated with miniproteins XMP-7, XMP-8, and XMP-34 showed that the enriched signal was not present in all designed miniproteins, including ones that share the same binding motifs as XMP-6 but have different scaffolds (e.g., XMP-7 and XMP-8).
  • FIG. 7A-B are images of fluorescent imaging of in vitro internalization of CA-IV- binding miniproteins of the invention.
  • Images show HEK293 cells incubated with miniproteins XMP-27, XMP-28, XMP-39, and XMP-52.
  • the intracellular fluorescent signal was enhanced only in permeabilized cells overexpressing CA-IV receptor, indicating potential internalization of the miniproteins.
  • HA-tagged mini-proteins were incubated with human LRP6 E1/E2 domain expressing HeLa cells at 2.5 pM for 1 hour at 37C. Immunostaining of supernatant and purified fractions was conducted.
  • FIG. 8A-B are images of fluorescent imaging of in vitro internalization of LY6A-binding miniproteins of the invention.
  • Selected miniproteins were expressed in E. coli and purified using a single step Ni-N A- based affinity purification.
  • An SPR assay was used to evaluate the binding interaction between receptor proteins and purified miniproteins. 200 nM Fc-tagged receptor protein were immobilized on a Protein A-precoated capture sensor, and engineered miniproteins at different concentrations were subsequently introduced.
  • FIG. 9A-F are graphs of SPR curves of purified miniproteins binders at different concentrations.
  • mice C57BL/6J mice (LY6A+) and BALB/cJ mice (LY6a-) were injected with 0.05mg HA-tagged miniprotein through retroorbital injection, and the tissues were harvested at indicated time before being fixed and stained for HA tags.
  • FIG. 10A-E show sagittal slices of tissues from mice injected with miniproteins of the invention.
  • images of sagittal slices showed LY6A-dependent XMP-6 enrichment across the brain in C57BL/6J mouse (LY6A+) but not in BALB/cJ mouse (LY6a-).
  • zoomed-in brain slice images indicated that XMP-6 distribution was enriched near brain vasculature.
  • zoomed-in brain slice images of XMP-28, a potential mouse CA- IV-binding miniprotein, showed enrichment near brain vasculature.
  • the miniproteins effectively bound to the BBB receptor and, when injected systemically, was enriched in the brain of LY6A-expressing mice. These receptor-binding miniproteins were shown to be potentially powerful tools to deliver therapeutics and diagnostic agents to specific organs.

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Abstract

The present invention provides compositions and methods comprising miniproteins as shuttles inducing transcytosis across target tissues, for example through an astrocyte-specific receptor, neuron-specific receptor, carbonic anhydrase IV (CA-IV), low density lipoprotein receptor related protein 6 (LRP6), or lymphocyte antigen 6 complex, locus A (LY6A).

Description

Compositions for Transport of Therapeutic Cargos Using Miniprotein Binders Targeting CA-IV
Government License Rights Statement
This invention was made with government support under Grant No. NS 111369 awarded by the National Institutes of Health. The government has certain rights in the invention.
Field of the Invention
The invention relates to methods and shuttles for crossing the blood brain barrier.
Background
The blood brain barrier (BBB) presents a fundamental bottleneck to the development of effective research tools and therapeutics for the central nervous system (CNS). This structure, comprising mainly of brain endothelial cells, requires large molecules to be delivered via invasive intracranial injections, technically challenging focused ultrasound, or receptor-mediated transcytosis. The rational design of BBB-crossing large molecules has long been hampered by the imperfect understanding of the mechanisms involved in transcytosis, with only a handful of targets, such as the transferrin receptor, validated for research and therapies.
Thus, the identification of BBB-crossing targets, mechanisms, molecules and methods is needed to improve the efficiencies of research tools and therapies for CNS.
Summary
The present invention provides compositions and methods comprising miniproteins as shuttles inducing transcytosis across target tissues, for example through an astrocyte-specific receptor, neuron-specific receptor, carbonic anhydrase IV (CA-IV), low density lipoprotein receptor related protein 6 (LRP6), or lymphocyte antigen 6 complex, locus A (LY6A, also known as stem cell antigen- 1 (SCA-1)).
Advantageously, the present invention provides miniproteins, typically between 5-20 kD in size, that can be modularly fused to various therapeutic cargos without disrupting functions of the cargos. Their epitope-informed design of miniproteins of the invention ensures they bind effectively to relevant sites. Coupled with a strengthened affinity through an expanded binding interface and favorable drug-like properties such as thermostability and high solubility, miniproteins represent a promising avenue for targeted therapeutic delivery. Further advantageously, miniproteins of the invention can be cost-effectively produced in E. coli.
Aspects of the invention provide a conjugate comprising a miniprotein shuttle which binds to a portion of a transcytosis-mediating cell receptor and a therapeutic cargo conjugated to the shuttle. Advantageously, the shuttle mediates transcytosis across the receptor, for example an astrocyte-specific receptor, neuron-specific receptor, CA-IV, LRP6, or LY6A.
Exemplary miniproteins are described in the table hereinbelow: Table 1: Select miniprotein sequences
For sequences disclosed throughout this application, it is understood nucleic acid molecules and peptides may comprise one or more substitutions, for example conservative substitutions, that allow sequences to continue to function. Accordingly, sequences may have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence disclosed.
A conservative substitution refers to amino acid substitutions that do not significantly affect or alter binding characteristics of a particular protein. Generally, conservative substitutions are ones in which a substituted amino acid residue is replaced with an amino acid residue having a similar side chain. For example, conservative substitutions may include a substitution found in one of the following groups: Group 1 : Alanine (Ala or A), Glycine (Gly or G), Serine (Ser or S), Threonine (Thr or T); Group 2: Aspartic acid (Asp or D), Glutamic acid (Glu or Z); Group 3: Asparagine (Asn or N), Glutamine (Gin or Q); Group 4: Arginine (Arg or R), Lysine (Lys or K), Histidine (His or H); Group 5: Isoleucine (He or I), Leucine (Leu or L), Methionine (Met or M), Valine (Vai or V); and Group 6: Phenylalanine (Phe or F), Tyrosine (Tyr or Y), Tryptophan (Trp or W). Additionally, or alternatively, amino acids can be grouped into conservative substitution groups by similar function, chemical structure, or composition (e.g., acidic, basic, aliphatic, aromatic, or sulfur-containing). For example, an aliphatic grouping may include, for purposes of substitution, Gly, Ala, Vai, Leu, and He. Other conservative substitutions groups include sulfur- containing: Met and Cysteine (Cys or C); acidic: Asp, Glu, Asn, and Gin; small aliphatic, nonpolar, or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; polar, negatively charged residues and their amides: Asp, Asn, Glu, and Gin; polar, positively charged residues: His, Arg, and Lys; large aliphatic, nonpolar residues: Met, Leu, He, Vai, and Cys; and large aromatic residues: Phe, Tyr, and Trp.
Accordingly, in aspects of the invention:
The miniprotein may bind the transcytosis-mediating cell receptor comprises a sequence having at least 95% sequence with a sequence selected from SEQ ID NO: 1-248.
The miniprotein may bind the transcytosis-mediating cell receptor LY6A and comprises a sequence having at least 95% sequence with a sequence selected from SEQ ID NO: 1-16, SEQ ID NO: 29-31, SEQ ID NO: 41, SEQ ID NO: 52, SEQ ID NO: 122, SEQ ID NO: 128-129, SEQ ID NO: 139-142, or SEQ ID NO: 144-147.
The miniprotein may bind the transcytosis-mediating cell receptor CA-IV and comprises a sequence having at least 95% sequence with a sequence selected from SEQ ID NO: 17-28, SEQ ID NO: 32-37, SEQ ID NO: 42-49, SEQ ID NO: 54-92, SEQ ID NO: 110-121, SEQ ID NO: 123-125, SEQ ID NO: 131-138, SEQ ID NO: 143, SEQ ID NO: 148, or SEQ ID NO: 171- 248.
The miniprotein may bind the transcytosis-mediating cell receptor LRP6 and comprises a sequence having at least 95% sequence with a sequence selected from SEQ ID NO: 38-40, SEQ ID NO: 44, SEQ ID NO: 53, SEQ ID NO: 93-109, SEQ ID NO: 126, SEQ ID NO: 130, SEQ ID NO: 149-170, or SEQ ID NO: 225-227.
The miniprotein may bind an astrocyte-specific or neuron-specific receptor and comprises a sequence having at least 95% sequence with a sequence selected from SEQ ID NO: 50-51 or SEQ ID NO 127.
The therapeutic cargo may be conjugated to the shuttle via a linker. Therapeutic cargo may covalently conjugated to the shuttle.
The therapeutic cargo may be a biological molecule, for example a nucleic acid (for example, RNA, siRNA, DNA, or an ASO), a protein (for example, an enzyme), a peptide, an antibody, a nanobody, a lipid, a polysaccharide, and a combination thereof.. The therapeutic cargo may be a non-biological molecule, for example a small molecule or a dye.
In aspects of the invention, the payload may comprise DNA molecules, oligonucleotides, therapeutic proteins, small molecule therapeutics, interfering RNA, gene editing cargo, chemotherapeutic agents, toxins, radioisotopes, enzymes, chelators, boron compounds, photoactive agents, dyes, metals, metal alloys, nanoparticles, or other larger synthetic molecules and biologies in vitro and in vivo.
The miniprotein may be in any known form, for example as described hereinbelow. For example, the miniprotein may be in a monomeric form, bi-specific form , multi-specific form, or as part of an alternative protein scaffolds. The miniprotein may be cystine-reinforced, hydrophobic core, or chemically-stabilized
Together, the conjugate may be characterized by delivery of the therapeutic cargo across the blood brain barrier (BBB). The therapeutic cargo may be therapeutic cargo for the treatment of a disorder affecting the central nervous system. For example, the conjugate may facilitate transcytosis across a target tissue including brain or eye tissue.
In aspects of the invention, miniproteins may also be provided with or without other protein scaffolds in other delivery systems, including viral vectors (c. ., lentivirus, adenovirus, AAVs), non-viral nanoparticles, exosomes, antibodies, antibody-drug conjugates, or proteins.
Aspects of the invention further provide methods and uses that comprise delivery of a therapeutic cargo across a transcytosis-mediating receptor of a subject. Methods and uses of the invention (including in the formulation of a medicament), comprise providing to a subject a conjugate of the invention, as described throughout this application, comprising a miniprotein shuttle of the invention and a therapeutic cargo conjugated to the shuttle.
Brief Description of the Drawings
FIG. 1 is a workflow for the design and screen of miniproteins of the invention.
FIG. 2A-B are images from Western blots from the synthesis of miniproteins of the invention. FIG. 3A-H are graphs of surface plasmon resonance (SPR) curves of LY6A-binding miniproteins of the invention.
FIG. 4AA-4BT are graphs of SPR curves of CA-IV-binding miniproteins of the invention.
FIG. 5A-5J are graphs of SPR curves of LRP6-binding miniproteins of the invention.
FIG. 6A-D are images of fluorescent imaging of in vitro internalization of LY6A-binding miniproteins of the invention.
FIG. 7A-B are images of fluorescent imaging of in vitro internalization of CA-IV- binding miniproteins of the invention.
FIG. 8A-B are images of fluorescent imaging of in vitro internalization of LY6A-binding miniproteins of the invention.
FIG. 9A-F are graphs of SPR curves of purified miniproteins binders at different concentrations.
FIG. 10A-E show sagittal slices of tissues from mice injected with miniproteins of the invention.
Detailed Description
The present invention provides compositions and methods comprising miniproteins as shuttles inducing transcytosis across target tissues, for example through an astrocyte-specific receptor, neuron-specific receptor, carbonic anhydrase IV (CA-IV), low density lipoprotein receptor related protein 6 (LRP6), or lymphocyte antigen 6 complex, locus A (LY6A).
Miniproteins
“Miniproteins,” “computationally designed binders,” “small-sized binders,” used interchangeably herein, are a diverse group of protein scaffolds characterized by small size, stability, and versatility in drug-like roles.
Advantageously, miniproteins may contain stable tertiary structures that facilitate specificity and potency in addition to resistance to proteolysis, reduction, and denaturation while also being of sufficiently small size that synthetic production, complete characterization, and regulation as a small molecule drug rather than as a biological product is possible. Their size also facilitates computational design and SAR as well as favorable penetration through tissue and manufacturing flexibility.
Some miniproteins can be segregated into three general subcategories, based on biophysical properties that impact their pharmacologic capabilities and biosynthetic production strategies: cystine-reinforced, hydrophobic core, and chemically-stabilized
The largest miniproteins are the hydrophobic core miniproteins, whose structure and stability are driven by rigid secondary structural elements (a-helix and 0-sheet) arranged around a hydrophobic core whose solvent exclusion drives its folding.
Examples of hydrophobic core miniproteins include affibodies, adnectin, centyrins, nanofittins, affitins, and funomers.
Adnectins and centyrins are based on fibronectin type 3 (10Fn3) domains, with similarities to antibody VH domains with prominent loops extending out from a quasi-P-barrel structure. Fynomers are adapted from the SH3 domain of Fyn kinase and are primarily P-barrels with exposed loops for binding. Affibodies are simple three-helix domains based on the antibody-engaging Z-domain of Protein A. Nanofittins and affitins are adapted from various 7- kDa DNA binding proteins from thermophilic archea and possess extraordinary thermal stability. All four classes have no need for chemical or oxidative stapling; as such, they can be produced in bacteria in large quantities. The binding of hydrophobic core miniproteins is mainly driven by similar strategies as in antibody Fv domains, comprising randomized sequences within loops or helices attached to a rigid superstructure.
Cystine-reinforced miniproteins use disulfides to drive folding and provide rigidity, with their folding and stability dependent on Cys-Cys disulfides.
Examples of cystine-reinformed miniproteins include avimers, kunitdomains, and cystine dense peptides (CDPs).
Cystine-dense peptides (CDPs) are represented by structurally diverse scaffolds that have similar biophysical properties, including native naturally cell-penetrant or blood-brain barrierpenetrant peptides. Advantageously, the striking protease resistance provided by the cystine knot can enable activity in aggressive environments such as the gastrointestinal (GI) tract. Kunitz domains have similarities to CDPs but contain a hydrophobic core and are specialized for protease inhibition; hundreds of such proteins exist in nature, such as the popular engraftment scaffold human APPI. Avimers are based on the loop-rich A-domains of human cell surface receptors, and, unlike CDPs or Kunitz domains, require coordination of a calcium ion. Avimers have been shown to be highly amenable to multimerization. While being smaller than hydrophobic core miniproteins, the screening strategies for cystine-reinforced miniproteins are similar to that of larger scaffolds. Binding is imparted by identifying surface exposed regions to engraft known binder motifs or randomized sequences, or by whole-protein (sparing cysteines) mutational screening.
Chemically stabilized miniproteins are the most similar to small molecule drugs in both size and functionality. Chemically stabilized miniproteins have unique properties but can roughly be considered as miniature, rigidified versions of 0-sheets (P-hairpins), a-helices (stapled peptides), or loops (bicycles) found at native protein-protein interfaces. All rely on a chemical crosslinking step to stabilize what would otherwise be unstable or unstructured peptide sequences.
Examples of chemically stabilized miniproteins include P-hairpins, stapled peptides, and bicycles.
Payload delivery across the receptors
Disclosed herein include methods and delivery systems for delivering a payload (e.g., a therapeutic agent) across a tissue surface via a transcytosis-mediating receptor, for example an astrocyte-specific receptor, neuron-specific receptor, CA-IV, LRP6, or LY6A.
The method comprises providing a miniprotein capable of interacting with an astrocytespecific receptor, neuron-specific receptor, CA-IV, LRP6, or LY6A. The miniprotein can be part of a delivery system and the delivery system can comprise a payload to be delivered to a nervous system. The method can further comprise administering the delivery system to the subject.
In some embodiments, the delivery system comprises nanoparticles, nanotubes, nanowires, dendrimers, liposomes, ethosomes and aquasomes, polymersomes and niosomes, foams, hydrogels, cubosomes, quantum dots, exosomes, macrophages, and combinations thereof. In some embodiments, the delivery system comprises a nanoparticle selected from lipid-based nanoparticles, polymeric nanoparticles, inorganic nanoparticles, surfactant-based emulsions, nanowires, silica nanoparticles, virus-like particles, peptide or protein-based particles, lipidpolymer particles, nanolipoprotein particles, and combinations thereof. For example, the payload may include an antimicrobial agent, a therapeutic agent, a prodrug, a peptide, a protein, an enzyme, a lipid, a biological response modifier, a pharmaceutical agent, a lymphokine, a heterologous antibody or fragment thereof, a detectable label, a polyethylene glycol (PEG) molecule, or a combination of two or more of the agents.
The payload can include a neuroactive polypeptide, for example, a neurotrophic factors, endocrine factors, growth factors, paracrine factors, hypothalamic release factors, neurotransmitter polypeptides, polypeptide agonists for a receptor expressed by a CNS cell, polypeptides involved in lysosomal storage disease or any combination thereof. In another example, the payload can include an IL-1 receptor antagonist (IL-IRa), dalargin, an interferon-P, Glial-derived neurotrophic factor (GDNF), tumor necrosis factor receptor (TNFR), nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin-4/5, neurotrophin (NT)-3, a neurturin, neuregulin, a netrin, ciliary neurotrophic factor (CNTF), stem cell factor (SCF), a semaphorin, hepatocyte growth factor (HGF), epidermal growth factor (EGF), transforming growth factor (TGF)-cx, TGF-B, vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), heregulin, artemin, persephin, interleukins, granulocyte-colony stimulating factor (CSF), granulocyte-macrophage-CSF, cardiotrophin-1, hedgehogs, leukemia inhibitory factor (LIF), midkine, pleiotrophin, erythropoietin (EPO), bone morphogenetic proteins (BMPs), netrins, saposins, any fragment thereof, or any combination thereof.
Aspects of the invention also provide for delivery of the conjugate to a subject in order to transport a therapeutic agent across the BBB. In aspects of the invention, delivery of the therapeutic payload may be for the treatment of a disease, disorder, or injury of the CNS. In aspects of the invention, the therapeutic agent may be released from the conjugate following entry into the CNS. In certain aspects, the disease, disorder, or injury of the CNS can be, without limitation, multiple sclerosis (MS), amylotrophic lateral sclerosis (ALS), Huntington's disease, Alzheimer's disease, Parkinson's disease, spinal cord injury, traumatic brain injury, stroke, neuropathic pain, neurodegeneration, neuroinflammation, progressive multifocal leukoencephalopathy (PML), encephalomyelitis (EPL), central pontine myelolysis (CPM), adrenoleukodystrophy, Alexander's disease, Pelizaeus Merzbacher disease (PMZ), Globoid cell Leucodystrophy (Krabbe's disease), Wallerian Degeneration, optic neuritis, transverse myelitis, post radiation injury, neurologic complications of chemotherapy, acute ischemic optic neuropathy, vitamin E deficiency, isolated vitamin E deficiency syndrome, Bassen-Kornzweig syndrome, Marchiafava-Bignami syndrome, metachromatic leukodystrophy, trigeminal neuralgia, Bell's palsy, primary tumors, secondary metastases, or any combination thereof.
Carbonic Anhydrase IV
The carbonic anhydrases (or carbonate dehydratases) (CAs) are a family of enzymes that catalyze the interconversion between carbon dioxide and water and the dissociated ions of carbonic acid. CAs participate in a variety of biological processes, including respiration, calcification, acid-base balance, bone resorption, and the formation of aqueous humor, cerebrospinal fluid, saliva, and gastric acid.
Carbonic anhydrase IV is an isozyme that belongs to the carbonic anhydrase family, a family of zinc metalloenzymes, which catalyzes the reversible reaction of hydration of CO2 (TBO+CCh^HCCh — I-H+), allowing the enzyme to regulate intra- and extra-cellular concentrations of CO2, H+, and HCO3 -. The carbonic anhydrases participate in a variety of biological processes, including respiration, calcification, acid-base balance, bone resorption, and the formation of aqueous humor, cerebrospinal fluid, saliva, and gastric acid. The carbonic anhydrases show extensive diversity in tissue distribution and in their subcellular localization. There are at least seven genetically distinct isozymes of mammalian carbonic anhydrase, designated I- VII, each of which catalyzes the reversible hydration of carbon dioxide by a zinchydroxide mechanism. Physiological functions that are regulated by carbonic anhydrase comprise, for example, removal of HCO3- in lung by respiration, reutilization of HCO3- in kidney, production of aqueous humor in eyes, cerebrospinal fluids in brain, gastric juice production in stomach, pancreatic juice, and bone resorption by osteoclasts. Carbonic anhydrase family members also play important roles in metabolic processes that include ureagenesis, gluconeogenesis, and lipogenesis.
Different from other carbonic anhydrases that are either soluble or attached to the plasma membrane by a membrane-spanning domain, carbonic anhydrase IV is a glycosylphosphatidylinositol-anchored membrane isozyme. Carbonic anhydrase IV is broadly conserved across vertebrates and has similar CNS expression profiles in humans, with a recent single cell analysis of human brain vasculature confirming CA-IV's expression in the human BBB. Carbonic anhydrase IV has been shown to regulate pH, which is associated with neural discharge and can influence neuronal function through ion-gated channels. In some embodiments, the carbonic anhydrase IV disclosed herein is a human carbonic anhydrase IV. CA-IV is known to localize on the luminal surface of brain endothelial cells throughout the cortex and cerebellum where it enzymatically modulates carbon dioxidebicarbonate balance. Human CA-IV has been previously characterized as a 35-kDa protein with a “high activity” in CO2 hydration and a higher activity than other isozymes in catalyzing the dehydration of HC03 -. In general, human CA-IV contains an 18-amino acid signal sequence at the N-terminal of the protein for endoplasmic reticulum (ER) translocation and a 260-amino acid “CA domain” containing active site amino acid residues that shows 30-36% homology with cytoplasmic CAs. At the C-terminal, an additional 27 amino acid residues containing the hydrophobic sequences of 21 amino acids sufficient to span the membrane are preceded by the 6- amino acid signal sequence for GPI-anchoring. The amino acid residue, Ser 266, was identified as the site for the attachment of the GPI anchor. The removal of C-terminal hydrophobic domain found in the CA-IV precursor has important impact on GPI-anchoring, cell surface expression, and realization of the enzyme activity. Based on amino acid sequences deduced from the nucleotide sequence, human CA-IV contains no classical consensus sites (Asn-Xxx-Ser/Thr) for N-glycosylation. Human CA-IV also contains no oligosaccharide chains, while other mammalian carbonic anhydrase IV (e.g. mouse carbonic anhydrases IV) are glycoproteins with one to several oligosaccharide side chains.
In some embodiments, the carbonic anhydrase IV disclosed herein is a mouse carbonic anhydrase IV. CA-IV has recently been found to be among the mouse proteins most strongly positively correlated with plasma-protein uptake in the brain (slightly stronger than the often- targeted transferrin receptor). This property is useful for identifying receptors for enhanced BBB crossing. CA-IV is also expressed in the GI tract, kidney, and lung, as well as taste receptor cells where it allows the sensing of carbonation. Mouse and human CA-IV are highly homologous, containing the same amino acids at positions crucial for enzyme activity (e.g., histidine residue 64 (His 64)), with several differences including, for example, that mouse CA-IV is an N-linked glycoprotein and the CO2 hydration rate catalyzed by mouse CA-IV is much lower than human CA-IV. Without being bound by any theory, the lower enzyme activity of mouse CA-IV may be associated with the replacement of Gly 63 in human CA-IV with Gin 63, among several other amino acid replacements. Another difference between mouse and human CA-IV is the Val-131- Asp-136 segment (13O's segment) that forms an a-helix in mouse and an extended loop in human CA-IV.
In some embodiments, a carbonic anhydrase IV disclosed herein as a receptor for enhancing BBB crossing can be any carbonic anhydrase IV, such as a mouse CA-IV, a human CA-IV, or a homology or a variant thereof. Carbonic anhydrase IV homologs and/or variants can be derived from a vertebrate species including, but not limited to, mouse, rat, human, bovine, rabbit, monkey, pig, horse, rainbow trout, chimpanzee, squirrel, chicken, goat, and sheep. Carbonic anhydrase IV homologs from various species can be found in public databases identifiable to a person skilled in the art, including for example UniProt, NCBI, and Swiss-Prot.
In some embodiments, a miniprotein can interact with a carbonic anhydrase IV disclosed herein (e.g., mouse CA-IV, human CA-IV or a homology or a variant thereof), thereby increasing permeability of the BBB (e.g., through transcytosis). In some embodiments, the increase in the permeability of the BBB is achieved by altering (e.g., increasing or decreasing) the carbonic anhydrase IV activity, such as reducing its activity.
In some embodiments, the alteration of carbonic anhydrase IV activity is achieved by a shuttle interacting to one or more active sites of the carbonic anhydrase IV including the zinc binding site and the hydrophobic substrate binding pocket. For example, the shuttle can interacting with the zinc binding site, the hydrophobic substrate binding pocket, or both.
The zinc binding site in carbonic anhydrase IV has a conserved structure dominated by a P-sheet super-structure with a metal binding site formed by at least three His residues. Without being bound by any theory, it is believed that the zinc binding site is on one face of the P-sheet at the bottom of a 15-A-deep, conical active site cleft in which zinc is liganded by three His residues and hydroxide ion with tetrahedral geometry. The hydrophobic substrate binding pocket is adjacent to zinc-bound hydroxide, formed in large part by bulky residues such as Vai at its base and Vai, Trp and Leu at its neck. This pocket is highly conserved among all active isozymes on the basis of phylogenetic comparisons. Without being bound by any theory, it is believed that the hydrophobic pocket has a minimum width and depth for efficient catalysis, and linear free energy relationships indicate that the volume of the amino acid residue at the base of the pocket and the hydrophobicity of residues at the neck of the pocket are critical for activity. Both the zinc binding site and the hydrophobic substrate binding pocket are highly conserved among carbonic anhydrase isozymes. low density lipoprotein receptor related protein 6 (LRP6)
The LRP6 gene encodes a member of the low density lipoprotein receptor (LDLR) gene family, which consists of cell surface proteins involved in receptor-mediated endocytosis of specific ligands. LDLR proteins are composed of the same basic structural motifs: an extracellular domain that contains LDLR binding repeats and EGF repeats with associated spacer domains containing the YWTD motif; a single transmembrane-spanning domain; and a C- terminal cytoplasmic domain that generally contains at least 1 copy of the NPXY motif.
Lrp6 is a single-pass transmembrane protein involved in the activation of Wnt signalling pathways. The human LRP6 has 23 exons, is found in chromosome 12p 13.2, and contains 1613 amino acids. LRP6 consists of four YWTD P-propeller domains, each of which is followed by an EGF-like domain, and this is followed by three LDLR type A repeats, a transmembrane domain, and a short intracellular domain. It is structurally related to LRP5, and they share almost 71% homology at the nucleotide level. In Lrp6, approximately 85% of the 1613-amino-acid length is extracellular. Most extracellular ligands bind LRP6 at the P-propellers. Each protein has a singlepass, 22-amino-acid segment that crosses the cell membrane and a 207-amino-acid segment that is internal to the cell. lymphocyte antigen 6 complex, locus A (LY6A)
Sca-1 is among the first identified members of the murine Ly6 gene family. The Ly6 gene family belongs to the superfamily of lymphocyte antigen-6 (Ly6)/urokinase-type plasminogen activator receptor (uPAR) proteins. This superfamily is characterized by the presence of LU domain. LU domain is a 60-80 amino acid domain, which is composed of 6-10 cysteines arranged in a specific spacing pattern that allows distinct disulfide bridges which create the three-fingered (3F) structural motif.
While a direct homolog of Sca-1 in humans is missing, human chromosome 8 — the syntenic region to mouse chromosome 15 — harbors several genes containing the characteristic s LU domain. The LU domain is found in extracellular domains of cell-surface receptors with membrane spanning domain (activin type 2 receptor and bone morphogenetic type IA receptor), or in GPI-anchored proteins CD 177 or in secreted globular proteins such CD59 antigen, SLURP 1/2. Experimental examples
Miniprotein binders were computational designed and tailored for specific receptors not only for crossing the blood brain barrier (BBB) but also for receptor-mediated delivery to other organs. The proprietary design methodology behind these miniproteins ensured they were epitope-informed, offering precise binding to targeted receptors. These designed miniproteins may enable specific and efficient therapeutic agent delivery.
Approximately 200 miniprotein binders were designed targeting different BBB receptors, such as mouse LY6A, human CA-IV, and human LRP6 and screened against their targets. It was shown using surface plasmon resonance (SPR) that miniproteins of the invention bind to their target receptors. Miniproteins were shown to further be internalized in cells overexpressing the target receptor. To demonstrate the in vivo application potential of the miniproteins binders, the in vivo distribution of a miniprotein targeting a BBB receptor found in rodent brains was characterized.
FIG. 1 is a three-stage workflow for the design and screen of miniproteins of the invention.
In stage 1, the receptor target was analyzed to formulate a design strategy: la) Binding motif identification: experimentally validated receptor binding motifs were identified by either literature or an in vitro pull-down selection; lb) Binding structure analysis: The complex structure of the binding motif and the receptor was obtained using either the protein data bank (PDB) database or predictive tools like AlphaFol d-Multimer; lc) Design strategy formation: A design strategy, such as epitope targeting or motif scaffolding, was determined based on available information.
In stage 2, miniproteins sequences in silica were design and evaluate:
2a) Backbone generation: Backbone structure models were generated using a diffusionbased generative Al, for example Rfdiffusion;
2b) Sequence design: The structure models were reverse-folded using tools like ProteinMPNN to generate protein sequences that likely fold into the desired structures; 2c) Design evaluation: The designed protein sequences were evaluated using AlphaFold confidence scores and APPRAISE ranking.
In stage 3, the designed sequences were screened and characterized:
3a) Cell-free protein synthesis: a small quantity of protein was synthesized using a cell- free protein production system;
3b) The produced protein was screened using surface plasmon resonance (SPR) or a cellbased binding assay;
3c) Single-protein characterization: hits from the screening were expressed in bacteria and subsequently assessed for their in vitro and in vivo binding, endocytosis, and transcytosis properties.
Cell-free design of miniproteins
Two batches of designed miniproteins were fused with HA tags and synthesized using NEB PureExpress, an E. COII.-'O^QA cell-free protein production system, following the manufacturer’s protocol. 1 microliter of each reaction was examined using Western blot for expression.
FIG. 2A-B are images from Western blots from the synthesis of miniproteins of the invention.
The blots were stained with 1 : 1000 anti-HA-HRP and bands were developed using UltraTMB substrate. Results indicated that the majority of designed miniproteins can be successfully produced to a concentration of 0.5-5 micromolar.
Miniprotein screen
200 nM of Fc-tagged LY6A receptor proteins were diluted in HBS-EF+ buffer with 1% BSA were immobilized on a Protein A chip. Miniproteins synthesized with the cell-free reactions, diluted in the same buffer as receptor proteins, were then flown to the chip.
FIG. 3A-P are graphs of SPR curves of LY6A-binding miniproteins of the invention.
The designed miniproteins, particularly the circled miniproteins (XMP-6, XMP-7, XMP- 34), showed binding signals. Note that the y-axis range and sample concentrations may differ between panels. 200 nM of Fc-tagged human CA-IV receptor proteins were diluted in HBS-EF+ buffer with 1% BSA were immobilized on a Protein A chip. Miniproteins synthesized with the cell-free reactions, diluted in the same buffer as receptor proteins, were then flown to the chip.
FIG. 4AA-4BT are graphs of SPR curves of CA-IV-binding miniproteins of the invention.
The designed miniproteins, particularly the circled miniproteins (XMP-27 and XMP-52), showed binding signals. Note that the y-axis range and sample concentrations may differ between panels.
200 nM of Fc-tagged human LRP6 receptor proteins were diluted in HBS-EF+ buffer with 1% BSA were immobilized on a Protein A chip. Miniproteins synthesized with the cell-free reactions, diluted in the same buffer as receptor proteins, were then flown to the chip.
FIG. 5A-5J are graphs of SPR curves of LRP6-binding miniproteins of the invention.
The designed miniproteins, particularly the circled miniproteins (XMP-42 and XMP-41), showed binding signals. Note that the y-axis range and sample concentrations may differ between panels.
In-vitro assays
In vitro internalization of designed LY6A-binding miniproteins was analyzed. Cultured HEK293 cells were transfected with receptor DNAs and incubated with 0.1 micromolar of HA- tagged miniproteins for Ihr before fixed with 4% PFA and, optionally, permeabilized with 0.3% Triton-XIOO. Cells were then stained with anti -HA primary antibody with fluorescent labeling and imaged.
FIG. 6A-D are images of fluorescent imaging of in vitro internalization of LY6A-binding miniproteins of the invention.
As shown in FIG. 6A, images of HEK293 cells incubated with miniproteins XMP-6, showed specific enrichment of XMP-6 in cells overexpressing LY6A receptor.
As shown in FIG. 6B, a Z-slice of representative cells showed internal granular signals that likely indicate endocytosis.
As shown in FIG. 6C, images of HEK293 cells incubated with miniproteins XMP-7, XMP-8, and XMP-34, showed that the enriched signal was not present in all designed miniproteins, including ones that share the same binding motifs as XMP-6 but have different scaffolds (e.g., XMP-7 and XMP-8).
In vitro internalization of designed CA-IV-binding miniproteins was analyzed. Cultured HEK293 cells were transfected with receptor DNAs and incubated with 0.1 micromolar of HA- tagged miniproteins for Ihr before fixed with 4% PFA and, optionally, permeabilized with 0.3% Triton-XIOO. Cells were then stained with anti -HA primary antibody with fluorescent labeling and imaged.
FIG. 7A-B are images of fluorescent imaging of in vitro internalization of CA-IV- binding miniproteins of the invention.
Images show HEK293 cells incubated with miniproteins XMP-27, XMP-28, XMP-39, and XMP-52. The intracellular fluorescent signal was enhanced only in permeabilized cells overexpressing CA-IV receptor, indicating potential internalization of the miniproteins.
In vitro internalization of designed LRP6-binding miniproteins XMP-184, XMP-185, and XMP-198 was analyzed.
HA-tagged mini-proteins were incubated with human LRP6 E1/E2 domain expressing HeLa cells at 2.5 pM for 1 hour at 37C. Immunostaining of supernatant and purified fractions was conducted.
FIG. 8A-B are images of fluorescent imaging of in vitro internalization of LY6A-binding miniproteins of the invention.
Selected miniproteins were expressed in E. coli and purified using a single step Ni-N A- based affinity purification. An SPR assay was used to evaluate the binding interaction between receptor proteins and purified miniproteins. 200 nM Fc-tagged receptor protein were immobilized on a Protein A-precoated capture sensor, and engineered miniproteins at different concentrations were subsequently introduced.
FIG. 9A-F are graphs of SPR curves of purified miniproteins binders at different concentrations.
Receptor concentrations are indicated in the inset. In vivo assay
In vivo characterization of designed mouse BBB receptor-binding miniproteins was conducted. C57BL/6J mice (LY6A+) and BALB/cJ mice (LY6a-) were injected with 0.05mg HA-tagged miniprotein through retroorbital injection, and the tissues were harvested at indicated time before being fixed and stained for HA tags.
FIG. 10A-E show sagittal slices of tissues from mice injected with miniproteins of the invention.
As shown in FIG. 10A, images of sagittal slices showed LY6A-dependent XMP-6 enrichment across the brain in C57BL/6J mouse (LY6A+) but not in BALB/cJ mouse (LY6a-).
As shown in FIG. 10B, images of livers showed that XMP-6 was present in the liver regardless of LY6A expression.
As shown in FIG. 10C-D, zoomed-in brain slice images indicated that XMP-6 distribution was enriched near brain vasculature.
As shown in FIG. 10E, zoomed-in brain slice images of XMP-28, a potential mouse CA- IV-binding miniprotein, showed enrichment near brain vasculature.
Conclusion
The miniproteins effectively bound to the BBB receptor and, when injected systemically, was enriched in the brain of LY6A-expressing mice. These receptor-binding miniproteins were shown to be potentially powerful tools to deliver therapeutics and diagnostic agents to specific organs.
Incorporation by Reference
References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, have been made throughout this disclosure. All such documents are hereby incorporated herein by reference in their entirety for all purposes.
Equivalents
Various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the full contents of this document, including references to the scientific and patent literature cited herein. The subject matter herein contains important information, exemplification and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents thereof.

Claims

Claims
1. A conjugate comprising: a shuttle comprising a miniprotein which binds to a portion of a transcytosis-mediating cell receptor; and a therapeutic cargo conjugated to the shuttle.
2. The conjugated of claim 1, wherein the transcytosis-mediating cell receptor is an astrocytespecific receptor, neuron-specific receptor, CA-IV, LRP6, or LY6A.
3. The conjugate of claim 1, wherein the miniprotein binds the transcytosis-mediating cell receptor comprises a sequence having at least 95% sequence with a sequence selected from SEQ ID NO: 1-248.
4. The conjugate of claim 1, wherein the miniprotein binds the transcytosis-mediating cell receptor LY6A and comprises a sequence having at least 95% sequence with a sequence selected from SEQ ID NO: 1-16, SEQ ID NO: 29-31, SEQ ID NO: 41, SEQ ID NO: 52, SEQ ID NO: 122, SEQ ID NO: 128-129, SEQ ID NO: 139-142, or SEQ ID NO: 144-147.
5. The conjugate of claim 1, wherein the miniprotein binds the transcytosis-mediating cell receptor CA-IV and comprises a sequence having at least 95% sequence with a sequence selected from SEQ ID NO: 17-28, SEQ ID NO: 32-37, SEQ ID NO: 42-49, SEQ ID NO: 54-92, SEQ ID NO: 110-121, SEQ ID NO: 123-125, SEQ ID NO: 131-138, SEQ ID NO: 143, SEQ ID NO: 148, or SEQ ID NO: 171-248.
6. The conjugate of claim 1, wherein the miniprotein binds the transcytosis-mediating cell receptor LRP6 and comprises a sequence having at least 95% sequence with a sequence selected from SEQ ID NO: 38-40, SEQ ID NO: 44, SEQ ID NO: 53, SEQ ID NO: 93-109, SEQ ID NO: 126, SEQ ID NO: 130, SEQ ID NO: 149-170, or SEQ ID NO: 225-227.
7. The conjugate of claim 1, wherein the miniprotein binds an astrocyte-specific or neuronspecific receptor and comprises a sequence having at least 95% sequence with a sequence selected from SEQ ID NO: 50-51 or SEQ ID NO 127.
8. The conjugate of claim 1, wherein the therapeutic cargo is conjugated to the shuttle via a linker.
9. The conjugate of claim 1, wherein the therapeutic cargo is covalently conjugated to the shuttle.
10. The conjugate of claim 1, wherein the therapeutic cargo is a biological molecule.
11. The conjugate of claim 10, wherein the biological molecule is selected from the group consisting of a nucleic acid, a protein, a peptide, an antibody, a nanobody, a lipid, a polysaccharide, and a combination thereof.
12. The conjugate of claim 1, wherein miniprotein is in a monomeric form, bi-specific form , multi-specific form, or as part of an alternative protein scaffolds.
13. The conjugate of claim 1, wherein the conjugate is characterized by delivery of the therapeutic cargo across the blood brain barrier (BBB).
14. The conjugate claim 13, wherein the therapeutic cargo is a therapeutic cargo for the treatment of a disorder affecting the central nervous system.
15. The conjugate of claim 14, wherein the target tissue is brain or eye tissue.
16. A method of delivering a therapeutic cargo across the tissue of a subject, the method comprising: providing to a subject a conjugate comprising: a shuttle comprising a miniprotein which binds to a portion of a transcytosis- mediating cell receptor; and a therapeutic cargo conjugated to the shuttle.
17. The method of claim 1, wherein the transcytosis-mediating cell receptor is an astrocytespecific receptor, neuron-specific receptor, CA-IV, LRP6, or LY6A.
18. The method of claim 1, wherein the miniprotein binds the transcytosis-mediating cell receptor comprises a sequence having at least 95% sequence with a sequence selected from SEQ ID NO: 1-248.
19. The method of claim 1, wherein the miniprotein binds the transcytosis-mediating cell receptor LY6A and comprises a sequence having at least 95% sequence with a sequence selected from SEQ ID NO: 1-16, SEQ ID NO: 29-31, SEQ ID NO: 41, SEQ ID NO: 52, SEQ ID NO: 122, SEQ ID NO: 128-129, SEQ ID NO: 139-142, or SEQ ID NO: 144-147.
20. The method of claim 1, wherein the miniprotein binds the transcytosis-mediating cell receptor CA-IV and comprises a sequence having at least 95% sequence with a sequence selected from SEQ ID NO: 17-28, SEQ ID NO: 32-37, SEQ ID NO: 42-49, SEQ ID NO: 54-92, SEQ ID NO: 110-121, SEQ ID NO: 123-125, SEQ ID NO: 131-138, SEQ ID NO: 143, SEQ ID NO: 148, or SEQ ID NO: 171-248.
21. The method of claim 1, wherein the miniprotein binds the transcytosis-mediating cell receptor LRP6 and comprises a sequence having at least 95% sequence with a sequence selected from SEQ ID NO: 38-40, SEQ ID NO: 44, SEQ ID NO: 53, SEQ ID NO: 93-109, SEQ ID NO: 126, SEQ ID NO: 130, SEQ ID NO: 149-170, or SEQ ID NO: 225-227.
22. The method of claim 1, wherein the miniprotein binds an astrocyte-specific or neuronspecific receptor and comprises a sequence having at least 95% sequence with a sequence selected from SEQ ID NO: 50-51 or SEQ ID NO 127.
23. The method of claim 1, wherein the therapeutic cargo is conjugated to the shuttle via a linker.
24. The method of claim 1, wherein the therapeutic cargo is covalently conjugated to the shuttle.
25. The method of claim 1, wherein the therapeutic cargo is a biological molecule.
26. The method of claim 10, wherein the biological molecule is selected from the group consisting of a nucleic acid, a protein, a peptide, an antibody, a nanobody, a lipid, a polysaccharide, and a combination thereof.
27. The method of claim 1, wherein miniprotein is in a monomeric form, bi-specific form , multi-specific form, or as part of an alternative protein scaffolds.
28. The method of claim 1, wherein the conjugate is characterized by delivery of the therapeutic cargo across the blood brain barrier (BBB).
29. The method claim 13, wherein the therapeutic cargo is a therapeutic cargo for the treatment of a disorder affecting the central nervous system.
30. The method of claim 14, wherein the target tissue is brain or eye tissue.
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