Composition for transporting therapeutic cargo using a miniprotein binder that targets CA-IV

Abstract

The present invention provides compositions and methods comprising miniproteins as shuttles for inducing transcytosis across target tissues via, for example, astrocyte-specific receptors, neuron-specific receptors, carbonic anhydrase IV (CA-IV), low-density lipoprotein receptor-associated protein 6 (LRP6), or lymphocyte antigen 6 complexes, or locus A (LY6A). Advantageously, the present invention provides miniproteins, typically between 5kD and 20kD in size, that can be modularly fused to various therapeutic cargoes without disrupting the cargo's function.

Description

Technical Field

[0001] Statement of Government License Rights This invention was made with government support under grant number NS111369 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0002] Field of the Invention The present invention relates to methods and shuttles for crossing the blood-brain barrier. Background Art

[0003] Background The blood-brain barrier (BBB) represents a fundamental bottleneck for the development of effective research tools and therapeutic agents for the central nervous system (CNS). This structure, which is mainly composed of brain endothelial cells, requires delivery of large molecules via invasive intracranial injection, technically challenging focused ultrasound, or receptor-mediated transcytosis. Rational design of BBB-crossing large molecules has long been hindered by incomplete understanding of the mechanisms involved in transcytosis, and only a small number of targets, such as the transferrin receptor, have been validated for research and therapy.

[0004] Accordingly, identification of BBB-crossing targets, mechanisms, molecules and methods is needed to improve the efficiency of research tools and therapies for the CNS. Summary of the Invention Means for Solving the Problems

[0005] Summary The present invention provides compositions and methods comprising, for example, astrocyte-specific receptors, neuron-specific receptors, carbonic anhydrase IV (CA-IV), low-density lipoprotein receptor-associated protein 6 (LRP6), or lymphocyte antigen 6 complex, and miniproteins as shuttles for inducing transcytosis across target tissues via locus A (LY6A, also known as stem cell antigen-1 (SCA-1)).

[0006] Advantageously, the present invention provides miniproteins, typically between 5 kD and 20 kD in size, that can be modularly fused to various therapeutic cargoes without disrupting the cargo's function. The epitope-informed design of the miniproteins of the present invention ensures that they efficiently bind to the appropriate sites. Coupled with enhanced affinity via extended binding interfaces and favorable drug-like properties, such as thermal stability and high solubility, the miniproteins present a promising avenue for targeted therapeutic delivery. Further advantageously, the miniproteins of the present invention can be produced cost-effectively in E. coli.

[0007] Aspects of the present invention provide a conjugate comprising a miniprotein shuttle that binds to a portion of a transcytosis-mediated cell receptor and a therapeutic cargo conjugated to the shuttle. Advantageously, the shuttle mediates transcytosis across the receptor, e.g., an astrocyte-specific receptor, a neuron-specific receptor, CA-IV, LRP6, or LY6A.

[0008] Exemplary miniproteins are listed in the table below: [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] Table 1-5 Table 1-6 Table 1-7 Table 1-8 Table 1-9 Table 1-10 Table 1-11 Table 1-12 Table 1-13 Table 1-14 Table 1-15 Table 1-16 Table 1-17 Table 1-18 Table 1-19 Table 1-20 Table 1-21 [Table 1-22] [Table 1-23] [Table 1-24] [Table 1-25] [Table 1-26] [Table 1-27] [Table 1-28] [Table 1-29] [Table 1-30] [Table 1-31] [Table 1-32] [Table 1-33] [Table 1-34]

[0009] With respect to the sequences disclosed through this application, it is understood that nucleic acid molecules and peptides may contain one or more substitutions, such as conservative substitutions, that allow the sequence to remain functional. Therefore, the sequences may have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to the disclosed sequences.

[0010] Conservative substitutions refer to amino acid substitutions that do not significantly affect or alter the binding characteristics of a particular protein. Generally, conservative substitutions are those in which the substituted amino acid residue is replaced by an amino acid residue with a similar side chain. For example, conservative substitutions may include substitutions 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 (Gln or Q); Group 4: arginine (Arg or R), lysine (Lys or K), histidine (His or H); Group 5: isoleucine (Ile or I), leucine (Leu or L), methionine (Met or M), valine (Val or V); and Group 6: phenylalanine (Phe or F), tyrosine (Tyr or Y), tryptophan (Trp or W). Furthermore, amino acids can be grouped into conserved substitution groups based on similar functions, chemical structures, or compositions (e.g., acidic, basic, aliphatic, aromatic, or sulfur-containing). For example, the aliphatic group may include Gly, Ala, Val, Leu, and Ile for substitution purposes. Other conserved substitution groups include sulfur-containing: Met and cysteine ​​(Cys or C); acidic: Asp, Glu, Asn, and Gln; small aliphatic, nonpolar, or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; polar, negatively charged residues and their amides: Asp, Asn, Glu, and Gln; polar, positively charged residues: His, Arg, and Lys; large aliphatic, nonpolar residues: Met, Leu, Ile, Val, and Cys; and large aromatic residues: Phe, Tyr, and Trp.

[0011] Therefore, in an embodiment of the present invention: The miniprotein can bind to transcytosis-mediated cell receptors and contains sequences selected from SEQ ID NOs: 1-248 and sequences having at least 95% of the sequence.

[0012] The miniprotein can bind to the transcytosis-mediated cell receptor LY6A and contains sequences selected from SEQ ID NOs: 1-16, SEQ ID NOs: 29-31, SEQ ID NOs: 41, SEQ ID NOs: 52, SEQ ID NOs: 122, SEQ ID NOs: 128-129, SEQ ID NOs: 139-142, or SEQ ID NOs: 144-147, and a sequence having at least 95% of the sequence.

[0013] The miniprotein can bind to the transcytosis-mediated cell receptor CA-IV and contains sequences selected from SEQ ID NOs: 17-28, 32-37, 42-49, 54-92, 110-121, 123-125, 131-138, 143, 148, or 171-248, and a sequence having at least 95% of the sequence.

[0014] The miniprotein can bind to the transcytosis-mediated cell receptor LRP6 and contains sequences selected from SEQ ID NOs: 38-40, SEQ ID NOs: 44, SEQ ID NOs: 53, SEQ ID NOs: 93-109, SEQ ID NOs: 126, SEQ ID NOs: 130, SEQ ID NOs: 149-170, or SEQ ID NOs: 225-227, and a sequence having at least 95% of the sequence.

[0015] The miniprotein can bind to astrocyte-specific receptors or neuron-specific receptors and contains a sequence selected from SEQ ID NOs. 50-51 or SEQ ID NO. 127, along with at least 95% of the sequence.

[0016] Therapeutic cargo can be conjugated to the shuttle via a linker. Therapeutic cargo can be covalently conjugated to the shuttle.

[0017] Therapeutic cargo can be biological molecules, such as nucleic acids (e.g., RNA, siRNA, DNA, or ASO), proteins (e.g., enzymes), peptides, antibodies, nanobodies, lipids, polysaccharides, or combinations thereof. Therapeutic cargo can also be non-biological molecules, such as small molecules or dyes.

[0018] In aspects of the present invention, the payload may include in vitro and in vivo DNA molecules, oligonucleotides, therapeutic proteins, small molecule therapeutic agents, 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 biologics.

[0019] The miniprotein may be any known form, as described below herein, for example. For example, the miniprotein may be in a monomeric form, a bispecific form, a polyspecific form, or as part of an alternative protein scaffold. The miniprotein may be reinforced with cystine, may be a hydrophobic core, or may be chemically stabilized.

[0020] In summary, conjugates can be characterized by the delivery of therapeutic cargo across the blood-brain barrier (BBB). Therapeutic cargo can be for the treatment of disorders affecting the central nervous system. For example, a conjugate may facilitate transcytosis across target tissues, including brain or ocular tissue.

[0021] In aspects of the present invention, miniproteins with or without other protein scaffolds in other delivery systems, including viral vectors (e.g., lentiviruses, adenoviruses, AAVs), nonviral nanoparticles, exosomes, antibodies, antibody-drug conjugates, or proteins, may also be provided.

[0022] Aspects of the present invention further provide methods and uses comprising the delivery of a therapeutic cargo across a target transcytosis-mediated receptor. Methods and uses of the present invention (including those in pharmaceutical formulation) include the step of providing a target conjugate of the present invention, comprising the miniprotein shuttle of the present invention and the therapeutic cargo conjugated to the shuttle, as described throughout this application. [Brief explanation of the drawing]

[0023] [Figure 1] Figure 1 shows the workflow for designing and screening the miniproteins of the present invention. [Figure 2A] Figures 2A and 2B are images from Western blots of the miniproteins synthesized according to the present invention. [Figure 2B] Same as above. [Figure 3A-B] Figures 3A-H show graphs of the surface plasmon resonance (SPR) curves of the LY6A-binding miniprotein of the present invention. [Figure 3C-D] Same as above. [Figure 3E-F] Same as above. [Figure 3G-H] Same as above. [Figure 3I-J] Same as above. [Figure 3K-L] Same as above. [Figure 3M-N] Same as above. [Figure 3O-P] Same as above. [Figure 4AA-AB] Figures 4AA to 4BT show graphs of the SPR curves of the CA-IV-binding miniprotein of the present invention. [Figure 4AC-AD] Same as above. [Figure 4AE-AF] Same as above. [Figure 4AG-AH] Same as above. [Figure 4AI-AJ] Same as above. [Figure 4AK-AL] Same as above. [Figure 4AM-AN] Same as above. [Figure 4AO-AP] Same as above. [Figure 4AQ-AR] Same as above. [Figure 4 AS-AT] Same as above. [Figure 4AU-AV] Same as above. [Figure 4AW-AX] Same as above. [Figure 4AY-AZ] Same as above. [Figure 4BA-BB] Same as above. [Figure 4BC-BD] Same as above. [Figure 4BE-BF] Same as above. [Figure 4BG-BH] Same as above. [Figure 4 BI-BJ]Same as above. [Figure 4BK-BL] Same as above. [Figure 4BM-BN] Same as above. [Figure 4BO-BP] Same as above. [Figure 4BQ-BR] Same as above. [Figure 4BS-BT] Same as above. [Figure 4BU] Same as above. [Figure 5A-B] Figures 5A to 5J show graphs of the SPR curves of the LRP6-binding miniprotein of the present invention. [Figure 5C-D] Same as above. [Figure 5E-F] Same as above. [Figure 5G] Same as above. [Figure 6A] Figures 6A-D show fluorescence imaging images of the in vitro internalization of the LY6A-binding miniprotein of the present invention. [Figure 6B] Same as above. [Figure 6C] Same as above. [Figure 6D] Same as above. [Figure 7A] Figures 7A and 7B show fluorescence imaging images of the in vitro internalization of the CA-IV binding miniprotein of the present invention. [Figure 7B] Same as above. [Figure 8A] Figures 8A and 8B show fluorescence imaging images of the in vitro internalization of the LY6A-binding miniprotein of the present invention. [Figure 8B] Same as above. [Figure 9A-B] Figures 9A-F show graphs of the SPR curves for purified miniprotein binders at different concentrations. [Figure 9C-D] Same as above. [Figure 9E-F] Same as above. [Figure 10A] Figures 10A-E show sagittal slices of tissue from mice injected with the miniprotein of the present invention. [Figure 10B] Same as above. [Figure 10C] Same as above. [Figure 10D] Same as above. [Figure 10E] Same as above. [Modes for carrying out the invention]

[0024] Detailed explanation The present invention provides compositions and methods comprising, for example, astrocyte-specific receptors, neuron-specific receptors, carbonic anhydrase IV (CA-IV), low-density lipoprotein receptor-associated protein 6 (LRP6), or lymphocyte antigen 6 complexes, and miniproteins as shuttles for inducing transcytosis across target tissues via locus A (LY6A).

[0025] Mini Protein The terms “miniproteins,” “computer-designed binders,” and “small-sized binders” used interchangeably herein refer to a diverse group of protein scaffolds characterized by their small size, stability, and versatility in drug-like roles.

[0026] Advantageously, miniproteins are small enough to be synthetically produced, fully characterized, and regulated as small-molecule drugs rather than biological products, while also potentially containing a stable tertiary structure that promotes specificity and potency, in addition to resistance to proteolysis, reduction, and denaturation. Their size also facilitates computer-aided design and SAR, as well as convenient penetration and manufacturing flexibility through tissues.

[0027] Some miniproteins can be separated into three general subcategories based on their pharmacological capabilities and biophysical properties that influence their biosynthetic production strategies: cystine reinforcement, hydrophobic core, and chemical stabilization.

[0028] The largest miniproteins are hydrophobic core miniproteins, whose structure and stability are driven by rigid secondary structural elements (α-helices and β-sheets) aligned around a hydrophobic core that drives their folding.

[0029] Examples of hydrophobic coremini proteins include affibody, adnectin, centyrin, nanofittin, affitin, and funomer.

[0030] Adnectin and centinlin are based on the fibronectin type 3 (10Fn3) domain, which has similarities to the antibody VH domain with an overhanging loop extending from a quasi-β-barrel structure. Fynomers are adapted from the SH3 domain of Fyn kinase and are primarily β-barrels with an exposed loop for binding. Affibodies are simple 3-helix domains based on the antibody-engaging Z-domain of protein A. Nanophytin and affitin are adapted from various 7kDa DNA-binding proteins derived from thermophilic archaea and possess extraordinary thermal stability. All four classes do not require either chemical or oxidative stapling; therefore, they can be produced in large quantities in bacteria. Binding of hydrophobic coreminiproteins is primarily driven by a strategy similar to that of the antibody Fv domain, involving randomized sequences within a loop or helix bound to a rigid superstructure.

[0031] Cystine-enhanced miniproteins use disulfides to drive folding and provide rigidity along with their folding and stability, which are dependent on Cys-Cys disulfides.

[0032] Examples of cystine-enhanced miniproteins include avimers, kunitdomains, and cystine-dense peptides (CDPs).

[0033] Cystine high-density peptides (CDPs) are represented by structurally diverse scaffolds with similar biophysical properties, including native, naturally occurring cell-transmissible or blood-brain barrier-transmissible peptides. Advantageously, the remarkable protease resistance provided by the cystine knot can enable activity in aggressive environments such as the gastrointestinal (GI) tract. Knitz domains have similarities to CDPs but contain a hydrophobic core and are specialized for protease inhibition; hundreds of such proteins exist naturally, including human APPI, a common engraftment scaffold. Avimers are based on the loop-rich A-domain of the human cell surface receptor and differ from both CDPs and Knitz domains, requiring calcium ion coordination. Avimers have been shown to be highly suitable for multimerization. Although smaller than hydrophobic core miniproteins, screening strategies for cystine-enhanced miniproteins are similar to those for larger scaffolds. Conjugation is achieved by identifying surface-exposed regions for engraving known binder motifs or randomized sequences, or by whole-protein (cysteine-preserving) mutation screening.

[0034] Chemically stabilized miniproteins are most similar to small molecule drugs in both size and functionality. While chemically stabilized miniproteins possess unique properties, they can be broadly considered as rigid miniature versions of β-sheets (β-hairpins), α-helices (stapled peptides), or loops (bicyclic structures) found at native protein-protein interfaces. All rely on a chemical cross-linking step to stabilize what would otherwise be an unstable or unstructured peptide sequence.

[0035] Examples of chemically stabilized miniproteins include β-hairpins, staple peptides, and bicyclic proteins.

[0036] Payload delivery across receptors Methods and delivery systems for delivering a payload (e.g., a therapeutic agent) across a tissue surface via transcytosis-mediated receptors, such as astrocyte-specific receptors, neuron-specific receptors, CA-IV, LRP6, or LY6A, are included in the disclosures herein.

[0037] This method includes the step of providing a miniprotein capable of interacting with astrocyte-specific receptors, neuron-specific receptors, CA-IV, LRP6, or LY6A. The miniprotein may be part of a delivery system, which may include a payload to be delivered to the nervous system. This method may further include the step of administering the delivery system to the target.

[0038] In some embodiments, the delivery system includes nanoparticles, nanotubes, nanowires, dendrimers, liposomes, ethosomes and aquasomes, polymerosomes and niosomes, foams, hydrogels, cubosomes, quantum dots, exosomes, macrophages, and combinations thereof. In some embodiments, the delivery system includes nanoparticles selected from lipid-based nanoparticles, polymeric nanoparticles, inorganic nanoparticles, surfactant-based emulsions, nanowires, silica nanoparticles, virus-like particles, peptide or protein-based particles, lipid-polymer particles, nanolipoprotein particles, and combinations thereof.

[0039] For example, the payload may include antibacterial agents, therapeutic agents, prodrugs, peptides, proteins, enzymes, lipids, biological response modifiers, pharmaceuticals, lymphokines, heterologous antibodies or fragments thereof, detectable labels, polyethylene glycol (PEG) molecules, or two or more combinations of these agents.

[0040] The payload may include neurotropic polypeptides, such as neurotrophic factors, endocrine factors, growth factors, paracrine factors, hypothalamic release factors, neurotransmitter polypeptides, polypeptide agonists for receptors expressed by CNS cells, polypeptides involved in lysosomal storage disorders, or any combination thereof. Another example of a payload is IL-1 receptor antagonist (IL-1Ra), dalargin, interferon-β, glial neurotrophic factor (GDNF), tumor necrosis factor receptor (TNFR), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-4 / 5, neurotrophin (NT)-3, neuruturin, neuregulin, netrin, ciliary neurotrophic factor (CNTF), stem cell factor (SCF), semaphorin, hepatocyte growth factor (HGF), epidermal growth factor (EGF), and transfer This may include forming growth factor (TGF)-cx, TGF-B, vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), heregulin, artemin, parcephin, interleukin, granulocyte colony-stimulating factor (CSF), granulocyte-macrophage-CSF, cardiotrophin-1, hedgehog, leukemia suppressor (LIF), midkine, pleiotrophin, erythropoietin (EPO), bone morphogenetic protein (BMP), netrin, saposin, any fragment thereof, or any combination thereof.

[0041] Aspects of the present invention also provide delivery of a conjugate to a target for transporting a therapeutic agent across the blood-brain barrier (BBB). In aspects of the present invention, the delivery of the therapeutic payload may be for the treatment of a disease, disorder, or injury of the central nervous system (CNS). In aspects of the present invention, the therapeutic agent may be released from the conjugate after entering the CNS. In certain aspects, diseases, disorders, or injuries of the CNS include, without limitation, multiple sclerosis (MS), amyotrophic 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 disease, Pelizaeus-Merzbach disease (PMZ), and globoid cell leukodystrophy. This may include leucodystrophy (Krabbe disease), Wallerian degeneration, optic neuritis, transverse myelitis, post-irradiation injury, neurological complications of chemotherapy, acute ischemic optic neuropathy, vitamin E deficiency, isolated vitamin E deficiency syndrome, Bassen-Kohnzweig syndrome, Marchia-Fava-Bignami syndrome, metachromatic leukodystrophy, trigeminal neuralgia, Bell's palsy, primary tumor, secondary metastasis, or any combination thereof.

[0042] Carbonic anhydrase IV Carbonic anhydrase (or dehydratase) (CA) is a family of enzymes that catalyze the interconversion of carbon dioxide and water with the dissociated ion of carbonic acid. CA is involved 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.

[0043] Carbonic anhydrase IV is responsible for the reversible hydration reaction of CO2 (H2O + CO2 ⇔ HCO3). - +H + This enzyme catalyzes CO2 and H + and HCO3 -Carbonic anhydrase is an isozyme belonging to the family of zinc metalloenzymes, which are capable of regulating intracellular and extracellular concentrations of carbon dioxide. Carbonic anhydrase is involved in various biological processes, including respiration, calcification, acid-base balance, bone resorption, and the formation of aqueous humor, cerebrospinal fluid, saliva, and gastric acid. Carbonic anhydrase exhibits a wide range of diversity in tissue distribution and their intracellular localization. Mammalian carbonic anhydrase has at least seven genetically distinct isozymes, called I-VII, each of which catalyzes the reversible hydration of carbon dioxide via the zinc-hydroxylase mechanism. Physiological functions regulated by carbonic anhydrase include, for example, the saturation of HCO3 in the lungs during respiration. - Removal of HCO3 in the kidneys - This includes the reuse of carbon dioxide, the production of aqueous humor in the eye, cerebrospinal fluid in the brain, gastric juice production in the stomach, pancreatic juice production, and bone resorption by osteoclasts. Carbonic anhydrase family members also play important roles in metabolic processes including urea production, gluconeogenesis, and lipid biosynthesis.

[0044] Unlike other carbonic anhydrases, which are either soluble or bound to the plasma membrane by a transmembrane domain, carbonic anhydrase IV is a membrane isozyme anchored with glycosylphosphatidyl-inositol. Carbonic anhydrase IV is widely conserved across vertebrates and has a similar CNS expression profile in humans, and recent single-cell analyses of human cerebral vascular structures have confirmed CA-IV expression in the human BBB. Carbonic anhydrase IV has been shown to regulate pH, which may be related to nerve discharge and affect neuronal function via ion gate channels.

[0045] In some embodiments, the carbonic anhydrase IV disclosed herein is human carbonic anhydrase IV. CA-IV is known to localize on the luminal surface of endothelial cells throughout the cortex and cerebellum, where it enzymatically modulates the carbon dioxide-bicarbonate balance. Human CA-IV has "high activity" in CO2 hydration and HCO3 -It has been previously characterized as a 35 kDa protein with higher activity than other isozymes in catalyzing dehydration. Generally, human CA-IV contains an 18-amino acid signal sequence at the N-terminus of the protein for endoplasmic reticulum (ER) translocation, and a 260-amino acid "CA domain" containing active site amino acid residues showing 30-36% homology with cytoplasmic CA. At the C-terminus, a further 27 amino acid residues containing a 21-amino acid hydrophobic sequence sufficient for transmembrane transport are preceded by a 6-amino acid signal sequence for GPI anchoring. Amino acid residue Ser266 was identified as the site for GPI anchor binding. Removal of the C-terminal hydrophobic domain found in CA-IV precursors has a significant impact on GPI anchoring, cell surface expression, and the realization of enzyme activity. Based on the amino acid sequence inferred from the nucleotide sequence, human CA-IV does not contain the classical consensus site (Asn-Xxx-Ser / Thr) for N-glycosylation. Human CA-IV does not contain oligosaccharide chains, whereas other mammalian carbonic anhydrase IV (e.g., mouse carbonic anhydrase IV) is a glycoprotein that has one to several oligosaccharide side chains.

[0046] In some embodiments, the carbonic anhydrase IV disclosed herein is mouse carbonic anhydrase IV. CA-IV has recently been found to be the mouse protein 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 cross-section. CA-IV is expressed in the GI tubules, kidneys and lungs, as well as in taste receptor cells, which enable it to sense carbonation. Mouse and human CA-IV are highly homologous and contain the same amino acid (e.g., histidine residue 64 (His64)) at a position crucial to enzymatic activity, with some differences including, for example, that mouse CA-IV is an N-linked glycoprotein and that the CO2 hydration rate catalyzed by mouse CA-IV is considerably lower than that of human CA-IV. While not bound by any theory, the lower enzymatic activity of mouse CA-IV may be related to the substitution of Gly63 with Gln63 in human CA-IV, among several other amino acid substitutions. Another difference between mouse CA-IV and human CA-IV is the Val-131 to Asp-136 segment (segments in the 130s), which forms an α-helix in mice and an extended loop in human CA-IV.

[0047] In some embodiments, the carbonic anhydrase IV disclosed herein as a receptor for enhancing BBB cross-section may be any carbonic anhydrase IV, e.g., mouse CA-IV, human CA-IV, or its homolog or variant. Carbonic anhydrase IV homologs and / or variants may originate from vertebrate species, including but not limited to mice, rats, humans, cattle, rabbits, monkeys, pigs, horses, rainbow trout, chimpanzees, squirrels, chickens, goats, and sheep. Carbonic anhydrase IV homologs from various species can be found in public databases identifiable to those skilled in the art, including, for example, UniProt, NCBI, and Swiss-Prot.

[0048] In some embodiments, the miniprotein can interact with carbonic anhydrase IV (e.g., mouse CA-IV, human CA-IV or its homolog or variant) disclosed herein, thereby increasing blood-brain barrier (BBB) ​​permeability (e.g., via transcytosis). In some embodiments, the increase in BBB permeability is achieved by altering (e.g., increasing or decreasing) the carbonic anhydrase IV activity, for example, by reducing its activity.

[0049] In some embodiments, modification of carbonic anhydrase IV activity is achieved by a shuttle that interacts with one or more active sites of carbonic anhydrase IV, including a zinc-binding site and a hydrophobic substrate-binding pocket. For example, the shuttle can interact with the zinc-binding site, the hydrophobic substrate-binding pocket, or both.

[0050] The zinc-binding site in carbonic anhydrase IV has a conserved structure governed by a β-sheet higher-order structure, with a metal-binding site formed by at least three His residues. While not bound by any particular theory, the zinc-binding site is thought to lie on one face of the β-sheet at the bottom of a 15 Å deep conical active site cleft where three His residues with tetrahedral geometry and hydroxide ions coordinate to zinc. The hydrophobic substrate-binding pocket is adjacent to the zinc-bound hydroxide and is largely formed by bulky residues, such as Val at its base and Val, Trp, and Leu at its neck. This pocket is highly conserved across all active isozymes based on phylogenetic comparisons. While not bound by any particular theory, the hydrophobic pocket has the minimum width and depth necessary for efficient catalysis, and linear free energy relationships suggest that the volume of amino acid residues at the base of the pocket and the hydrophobicity of residues at the neck of the pocket are important for activity. Both the zinc binding site and the hydrophobic substrate binding pocket are highly conserved among carbonic anhydrase isozymes.

[0051] 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 following identical basal structural motifs: an extracellular domain containing an EGF repeat with an associated spacer domain containing an LDLR-binding repeat and a YWTD motif; a single-pass transmembrane domain; and a C-terminal cytoplasmic domain generally containing at least one copy of the NPXY motif.

[0052] Lrp6 is a single-pass transmembrane protein involved in the activation of the Wnt signaling pathway. Human LRP6 has 23 exons, is found on chromosome 12p13.2, and contains 1613 amino acids. LRP6 consists of four YWTD β-propeller domains, each followed by an EGF-like domain, followed by three LDLR A-type repeats, a transmembrane domain, and a short intracellular domain. It is structurally related to LRP5, which shares nearly 71% homology at the nucleotide level. In Lrp6, approximately 85% of the 1613 amino acid length is extracellular. Most extracellular ligands bind to LRP6 at the β-propeller. Each protein has a single-pass transmembrane 22-amino acid segment that crosses the cell membrane and an internal 207-amino acid segment relative to the cell.

[0053] Lymphocyte antigen 6 complex, gene locus A (LY6A) Sca-1 is among the first identified members of the mouse 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 an LU domain. The LU domain is a 60-80 amino acid domain consisting of 6-10 cysteine ​​molecules aligned in a specific spacing pattern, enabling distinct disulfide crosslinks that create a three-finger (3F) structural motif.

[0054] While no direct homolog of Sca-1 has been found in humans, human chromosome 8—a synteny region relative to mouse chromosome 15—contains several genes that include a characteristic LU domain. The LU domain is found in the extracellular domains of cell-surface receptors with transmembrane domains (activin type 2 receptor and osteogenic receptor type IA), in the GPI anchoring protein CD177, or in secreted globular proteins, such as the CD59 antigen and SLURP1 / 2. [Examples]

[0055] Experimental example Mini-protein binders were computer-designed and tailored to 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 mini-proteins ensured they were based on epitope information, providing precise binding to targeted receptors. These engineered mini-proteins may enable specific and efficient therapeutic agent delivery.

[0056] Approximately 200 miniprotein binders targeting different BBB receptors, such as mouse LY6A, human CA-IV, and human LRP6, were designed and screened against their targets. Surface plasmon resonance (SPR) was used to demonstrate that the miniproteins of this invention bind to their target receptors. Further, it was shown that the miniproteins are internalized in cells overexpressing the target receptors. To demonstrate the in vivo applicability of the miniprotein binders, the in vivo distribution of miniproteins targeting BBB receptors found in rodent brains was characterized.

[0057] Figure 1 shows a three-step workflow for designing and screening the miniproteins of the present invention.

[0058] In Stage 1, we analyzed receptor targets and formulated a design strategy: 1a) Binding motif identification: Experimentally validated receptor binding motifs were identified by either literature review or in vitro pull-down selection; 1b) Binding structure analysis: The complex structures of the binding motif and receptor were obtained using either the Protein Data Bank (PDB) database or a predictive tool, such as AlphaFold-Multimer; 1c) Formulation of design strategy: A design strategy, such as epitope targeting or motif scaffolding, was determined based on the available information.

[0059] In stage 2, we designed and evaluated in silico miniprotein sequences: 2a) Skeleton generation: The skeletal structure model was generated using a diffusion-based generative AI, e.g., Rfdiffusion; 2b) Sequence design: The structural model was reverse-folded using tools such as ProteinMPNN to generate protein sequences that were likely to fold into the desired structure; 2c) Design evaluation: The designed protein sequences were evaluated using AlphaFold confidence scores and APPRAISE rankings.

[0060] In stage 3, the designed sequences were screened and characterized: 3a) Cell-free protein synthesis: Small amounts of protein were synthesized using a cell-free protein production system; 3b) The produced proteins were screened using surface plasmon resonance (SPR) or cell-based binding assays; 3c) Single protein characterization: Hits from the screening were expressed in bacteria, and subsequently evaluated for their in vitro and in vivo binding, endocytosis, and transcytosis properties.

[0061] Cell-free design of mini proteins Two batches of the designed miniproteins were fused with HA tags and synthesized using the E. coli-based cell-free protein production system NEB PureExpress according to the manufacturer's protocol. Each 1 microliter reaction was tested for expression using Western blotting.

[0062] Figures 2A and 2B are images from Western blots of the miniproteins synthesized according to the present invention.

[0063] The blots were stained with 1:1000 anti-HA-HRP, and the bands were color-developed using UltraTMB substrate. The results showed that the majority of the designed miniproteins could be successfully produced at concentrations of 0.5–5 micromoles.

[0064] Mini Protein Screening A 200 nM Fc-tagged LY6A receptor protein was diluted in HBS-EF+ buffer containing 1% BSA and immobilized on a Protein A chip. Then, miniproteins synthesized using a cell-free reaction, diluted in the same buffer as the receptor protein, were transferred to the chip.

[0065] Figures 3A-3P show graphs of the SPR curves of the LY6A-binding miniprotein of the present invention.

[0066] The designed mini-proteins, particularly the mini-proteins circled (XMP-6, XMP-7, XMP-34), showed binding signals. Note that the y-axis range and sample concentrations may differ between panels.

[0067] A 200 nM Fc-tagged human CA-IV receptor protein was diluted in HBS-EF+ buffer containing 1% BSA and immobilized on a Protein A chip. Then, miniproteins synthesized using a cell-free reaction, diluted in the same buffer as the receptor protein, were transferred to the chip.

[0068] Figures 4AA to 4BT show graphs of the SPR curves of the CA-IV-binding miniprotein of the present invention.

[0069] The designed mini-proteins, particularly the circled mini-proteins (XMP-27 and XMP-52), showed binding signals. Note that the y-axis range and sample concentrations may differ between panels.

[0070] A 200 nM Fc-tagged human LRP6 receptor protein was diluted in HBS-EF+ buffer containing 1% BSA and immobilized on a Protein A chip. Then, miniproteins synthesized using a cell-free reaction, diluted in the same buffer as the receptor protein, were transferred to the chip.

[0071] Figures 5A to 5J show graphs of the SPR curves of the LRP6-binding miniprotein of the present invention.

[0072] The designed mini-proteins, particularly the circled mini-proteins (XMP-42 and XMP-41), showed binding signals. Note that the y-axis range and sample concentrations may differ between panels.

[0073] in vitro assay The in vitro internalization of the designed LY6A-binding miniprotein was analyzed. Cultured HEK293 cells were transfected with receptor DNA and incubated with 0.1 micromolar concentration of HA-tagged miniprotein for 1 hour, then fixed with 4% PFA and permeabilized with 0.3% Triton®-X100 as needed. The cells were then stained with a fluorescently labeled anti-HA primary antibody and imaged.

[0074] Figures 6A-D show fluorescence imaging images of the in vitro internalization of the LY6A-binding miniprotein of the present invention.

[0075] As shown in Figure 6A, images of HEK293 cells incubated with the miniprotein XMP-6 showed specific enrichment of XMP-6 in cells overexpressing the LY6A receptor.

[0076] As shown in Figure 6B, Z-slices of representative cells showed internal granular signals that were likely indicative of endocytosis.

[0077] As shown in Figure 6C, images of HEK293 cells incubated with miniproteins XMP-7, XMP-8, and XMP-34 showed that enriched signals were absent for all designed miniproteins, including those that share the same binding motif as XMP-6 but have different scaffolds (e.g., XMP-7 and XMP-8).

[0078] The in vitro internalization of the designed CA-IV-binding miniprotein was analyzed. Cultured HEK293 cells were transfected with receptor DNA and incubated with 0.1 micromolar concentration of HA-tagged miniprotein for 1 hour, then fixed with 4% PFA and permeabilized with 0.3% Triton-X100 as needed. The cells were then stained with a fluorescently labeled anti-HA primary antibody and imaged.

[0079] Figures 7A and 7B show fluorescence imaging images of the in vitro internalization of the CA-IV binding miniprotein of the present invention.

[0080] The image shows HEK293 cells incubated with the miniproteins XMP-27, XMP-28, XMP-39, and XMP-52. Intracellular fluorescence signaling is enhanced only in permeabilized cells overexpressing the CA-IV receptor, indicating potential internalization of the miniproteins.

[0081] We analyzed the in vitro internalization of the designed LRP6-binding miniproteins XMP-184, XMP-185, and XMP-198.

[0082] HA-tagged miniproteins were incubated with human LRP6 E1 / E2 domain-expressing HeLa cells at 2.5 μM at 37°C for 1 hour. Immunostaining was performed on the supernatant and purified fractions.

[0083] Figures 8A and 8B show fluorescence imaging images of the in vitro internalization of the LY6A-binding miniprotein of the present invention.

[0084] The selected miniproteins were expressed in E. coli and purified using a single-step Ni-NTA-based affinity purification. The binding interaction between the receptor protein and the purified miniproteins was evaluated using an SPR assay. 200 nM Fc-tagged receptor protein was immobilized on a pre-coated capture sensor with protein A, and manipulated miniproteins at different concentrations were subsequently introduced.

[0085] Figures 9A-F show graphs of the SPR curves for purified miniprotein binders at different concentrations.

[0086] Receptor concentrations are shown in the inset diagram.

[0087] in vivo assay In vivo characterization of the designed mouse BBB receptor-binding miniprotein was performed. C57BL / 6J mice (LY6A+) and BALB / cJ mice (LY6a-) were injected with 0.05 mg of HA-tagged miniprotein via postorbital injection. Tissue was collected at indicated time points, then fixed and stained for the HA tag.

[0088] Figures 10A-E show sagittal slices of tissue from mice injected with the miniprotein of the present invention.

[0089] As shown in Figure 10A, sagittal slice images showed LY6A-dependent XMP-6 enrichment throughout the brain in C57BL / 6J mice (LY6A+), but not in BALB / cJ mice (LY6a-).

[0090] As shown in Figure 10B, liver images indicated that XMP-6 was present in the liver regardless of LY6A expression.

[0091] As shown in Figures 10C-D, zoomed-in brain slice images revealed that the XMP-6 distribution was enriched near vascular structures.

[0092] As shown in Figure 10E, zoomed-in brain slice images of XMP-28, a potential mouse CA-IV-binding miniprotein, showed enrichment near cerebral vascular structures.

[0093] conclusion The miniproteins efficiently bound to the BBB receptor and, when injected systemically, enriched the brains of LY6A-expressing mice. These receptor-binding miniproteins have been shown to be potentially powerful tools for delivering therapeutic and diagnostic agents to specific organs.

[0094] Import by reference References and citations to other materials, such as patents, patent applications, patent publications, journals, books, articles, and web content, have been made throughout this disclosure. All such materials are incorporated herein by reference in their entirety for all purposes.

[0095] Equal portions Various modifications of the present invention and its many further embodiments will be apparent to those skilled in the art from the entirety of this material, including references to scientific and patent documents cited herein, in addition to those shown and described herein. The subject matter of this specification includes important information, examples, and guidance that may be adapted for the implementation of the invention in its various embodiments and equivalents.

Claims

1. A shuttle containing a miniprotein that binds to a portion of the transcytosis-mediated cell receptor; and The therapeutic cargo conjugated to the aforementioned shuttle A conjugate that includes this.

2. The conjugate according to claim 1, wherein the transcytosis-mediated cell receptor is an astrocyte-specific receptor, a neuron-specific receptor, CA-IV, LRP6, or LY6A.

3. The conjugate according to claim 1, wherein the miniprotein binds to the transcytosis-mediated cell receptor and comprises a sequence having at least 95% of the sequence selected from SEQ ID NOs: 1 to 248.

4. The conjugate according to claim 1, wherein the miniprotein binds to the transcytosis-mediated cell receptor LY6A and comprises a sequence having at least 95% of the sequence selected from SEQ ID NOs: 1-16, SEQ ID NOs: 29-31, SEQ ID NOs: 41, SEQ ID NOs: 52, SEQ ID NOs: 122, SEQ ID NOs: 128-129, SEQ ID NOs: 139-142, or SEQ ID NOs: 144-147.

5. The conjugate according to claim 1, wherein the miniprotein binds to the transcytosis-mediated cell receptor CA-IV and comprises a sequence having at least 95% of the sequence selected from SEQ ID NOs: 17-28, 32-37, 42-49, 54-92, 110-121, 123-125, 131-138, 143, 148, or 171-248.

6. The conjugate according to claim 1, wherein the miniprotein binds to the transcytosis-mediated cell receptor LRP6 and comprises a sequence having at least 95% of the sequence selected from SEQ ID NOs. 38-40, SEQ ID NOs. 44, SEQ ID NOs. 53, SEQ ID NOs. 93-109, SEQ ID NOs. 126, SEQ ID NOs. 130, SEQ ID NOs. 149-170, or SEQ ID NOs. 225-227.

7. The conjugate according to claim 1, wherein the miniprotein binds to an astrocyte-specific receptor or a neuron-specific receptor and comprises a sequence having at least 95% of the sequence selected from SEQ ID NOs. 50-51 or SEQ ID NO.

127.

8. The conjugate according to claim 1, wherein the therapeutic cargo is conjugated to the shuttle via a linker.

9. The conjugate according to claim 1, wherein the therapeutic cargo is covalently conjugated to the shuttle.

10. The conjugate according to claim 1, wherein the therapeutic cargo is a biological molecule.

11. The conjugate according to claim 10, wherein the biological molecule is selected from the group consisting of nucleic acids, proteins, peptides, antibodies, nanobodies, lipids, polysaccharides, and combinations thereof.

12. The conjugate according to claim 1, wherein the miniprotein is in a monomeric form, a bispecific form, a polyspecific form, or as part of an alternative protein scaffold.

13. The conjugate according to claim 1, characterized by the delivery of the therapeutic cargo across the blood-brain barrier (BBB).

14. The conjugate according to claim 13, wherein the therapeutic cargo is a therapeutic cargo for the treatment of a disorder affecting the central nervous system.

15. The conjugate according to claim 14, wherein the target tissue is brain or eye tissue.

16. A method for delivering therapeutic cargo across target organizations, A shuttle containing a miniprotein that binds to a portion of the transcytosis-mediated cell receptor; and The therapeutic cargo conjugated to the aforementioned shuttle A method that includes steps to provide for a conjugate that includes

17. The method according to claim 1, wherein the transcytosis-mediated cell receptor is an astrocyte-specific receptor, a neuron-specific receptor, CA-IV, LRP6, or LY6A.

18. The method according to claim 1, wherein the miniprotein binds to the transcytosis-mediated cell receptor and comprises a sequence having at least 95% of the sequence selected from SEQ ID NOs: 1 to 248.

19. The method according to claim 1, wherein the miniprotein binds to the transcytosis-mediated cell receptor LY6A and comprises a sequence having at least 95% of the sequence selected from SEQ ID NOs: 1-16, SEQ ID NOs: 29-31, SEQ ID NOs: 41, SEQ ID NOs: 52, SEQ ID NOs: 122, SEQ ID NOs: 128-129, SEQ ID NOs: 139-142, or SEQ ID NOs: 144-147.

20. The method according to claim 1, wherein the miniprotein binds to the transcytosis-mediated cell receptor CA-IV and comprises a sequence having at least 95% of the sequence selected from SEQ ID NOs: 17-28, 32-37, 42-49, 54-92, 110-121, 123-125, 131-138, 143, 148, or 171-248.

21. The method according to claim 1, wherein the miniprotein binds to the transcytosis-mediated cell receptor LRP6 and comprises a sequence having at least 95% of the sequence selected from SEQ ID NOs. 38-40, SEQ ID NOs. 44, SEQ ID NOs. 53, SEQ ID NOs. 93-109, SEQ ID NOs. 126, SEQ ID NOs. 130, SEQ ID NOs. 149-170, or SEQ ID NOs. 225-227.

22. The method according to claim 1, wherein the miniprotein binds to an astrocyte-specific receptor or a neuron-specific receptor and comprises a sequence having at least 95% of the sequence selected from SEQ ID NOs. 50-51 or SEQ ID NO.

127.

23. The method according to claim 1, wherein the therapeutic cargo is conjugated to the shuttle via a linker.

24. The method according to claim 1, wherein the therapeutic cargo is covalently conjugated to the shuttle.

25. The method according to claim 1, wherein the therapeutic cargo is a biological molecule.

26. The method according to claim 10, wherein the biological molecule is selected from the group consisting of nucleic acids, proteins, peptides, antibodies, nanobodies, lipids, polysaccharides, and combinations thereof.

27. The method according to claim 1, wherein the miniprotein is in a monomeric form, a bispecific form, a polyspecific form, or as part of an alternative protein scaffold.

28. The method according to claim 1, wherein the conjugate is characterized by the delivery of the therapeutic cargo across the blood-brain barrier (BBB).

29. The method according to claim 13, wherein the therapeutic cargo is a therapeutic cargo for the treatment of a disorder affecting the central nervous system.

30. The method according to claim 14, wherein the target tissue is brain or eye tissue.

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