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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