Compositions for use in transporting therapeutic cargo using a small protein binder targeting ca-iv

Abstract

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

Description

[0001] Government Rights Statement This invention was carried out with government support under license number NS111369 granted by the National Institutes of Health. The government owns certain rights in this invention. Technical Field

[0002] This invention relates to methods and shuttles for crossing the blood-brain barrier. Background Technology

[0003] The blood-brain barrier (BBB) ​​is a fundamental bottleneck in the development of effective central nervous system (CNS) research tools and therapies. This structure primarily comprises brain endothelial cells, necessitating the delivery of macromolecules via invasive intracranial injection, technically challenging focused ultrasound, or receptor-mediated transcytosis. For a long time, an incomplete understanding of the mechanisms involved in transcytosis has hindered the rational design of macromolecules across the BBB, with only a few targets, such as the transferrin receptor, validated for research and therapies.

[0004] Therefore, it is necessary to identify targets, mechanisms, molecules and methods across the BBB to improve the efficiency of research tools and therapies in the CNS. Summary of the Invention

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

[0006] Advantageously, the present invention provides small proteins, typically ranging in size from 5 to 20 kDa, which can be modularly fused with a variety of therapeutic cargoes without compromising cargo functionality. The epitope-informed design of the small proteins of the present invention ensures their efficient binding to relevant sites. Combined with enhanced affinity through extended binding interfaces and favorable drug-like properties such as thermal stability and high solubility, small proteins represent a promising pathway for targeted therapeutic delivery. Further advantageously, the small proteins of the present invention can be produced economically and efficiently in *E. coli*.

[0007] Aspects of the present invention provide a conjugate comprising a small protein shuttle and a therapeutic cargo, the small protein shuttle binding to a portion of a cellular receptor mediating transcytosis, the therapeutic cargo being conjugated to the shuttle. Advantageously, the shuttle mediates transcytosis across a receptor, such as an astrocyte-specific receptor, a neuron-specific receptor, CA-IV, LRP6, or LY6A.

[0008] The following table describes exemplary small proteins: Table 1: Selected small protein sequences

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

[0010] "Conservative substitution" refers to amino acid substitutions that do not significantly affect or alter the binding properties of a specific protein. Typically, a conservative substitution is a substitution in which the substituted amino acid residue is replaced by an amino acid residue with a similar side chain. For example, conservative substitutions can 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). Alternatively, amino acids can be classified into conserved substitution groups based on similar functions, chemical structures, or compositions (e.g., acidic, basic, aliphatic, aromatic, or sulfur-containing). For example, for substitution purposes, the aliphatic group can include Gly, Ala, Val, Leu, and Ile. 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; negatively charged residues and their amides: Asp, Asn, Glu, and Gln; 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 various aspects of the present invention: The small protein can bind to cell receptors that mediate transcytosis, and the small protein comprises a sequence having at least 95% sequence content of a sequence selected from SEQ ID NO:1-248.

[0012] The small protein can bind to the cell receptor LY6A that mediates transcytosis and contains a sequence having at least 95% sequence content of a sequence selected from the following: 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.

[0013] The small protein can bind to the cell receptor CA-IV, which mediates transcytosis, and contains a sequence having at least 95% sequence content of a sequence selected from the following: 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.

[0014] The small protein can bind to the cell receptor LRP6, which mediates transcytosis, and contains a sequence having at least 95% sequence content of a sequence selected from the following: 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.

[0015] The small protein can bind to astrocyte-specific or neuron-specific receptors and includes a sequence having at least 95% sequence content of a sequence selected from SEQ ID NO: 50-51 or SEQ ID NO: 127.

[0016] Therapeutic cargo can be coupled to the shuttle via a joint. Therapeutic cargo can be covalently coupled to the shuttle.

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

[0018] In various aspects of the present invention, the payload may include DNA molecules, oligonucleotides, therapeutic proteins, small molecule therapeutic agents, interfering RNA, gene-editing cargo, chemotherapeutic agents, toxins, radioisotopes, enzymes, chelating agents, boron compounds, photosensitizers, dyes, metals, metal alloys, nanoparticles, or other larger synthetic molecules and biological agents in vitro and in vivo.

[0019] Small proteins can exist in any known form, such as those described below. For example, small proteins can be monomeric, bispecific, multispecific, or as part of an alternative protein scaffold. Small proteins can be cysteine-enhanced, hydrophobic, or chemically stable.

[0020] A common characteristic of conjugates is their ability to deliver therapeutic goods across the blood-brain barrier (BBB). These therapeutic goods can be used to treat conditions affecting the central nervous system. For example, conjugates can promote transcytosis across target tissues, including brain or eye tissues.

[0021] In various aspects of the invention, the small protein may also be provided 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.

[0022] Various aspects of the present invention further provide methods and uses for receptor delivery of therapeutic goods comprising mediated transcytosis across a subject. The methods and uses of the present invention (including in formulations of pharmaceuticals) comprise providing a subject with a conjugate of the present invention, as described throughout this application, the conjugate comprising a small protein shuttle of the present invention and a therapeutic goods conjugated to the shuttle. Attached Figure Description

[0023] Figure 1 This is the workflow for the design and screening of small proteins according to the present invention.

[0024] Figure 2A -B is an image of the protein blot of the synthesis of the small protein of the present invention.

[0025] Figure 3A -H is a graph of the surface plasmon resonance (SPR) curve of LY6A binding to the small protein of the present invention.

[0026] Figure 4AA-4BT This is a graph showing the SPR curve of the CA-IV binding small protein of the present invention.

[0027] Figure 5A -5J is a graph showing the SPR curve of LRP6 binding to small proteins in this invention.

[0028] Figure 6A -D is a fluorescence imaging image of the in vitro internalization of the LY6A-bound small protein of the present invention.

[0029] Figure 7A -B is a fluorescence imaging image of the in vitro internalization of the CA-IV-binding small protein of the present invention.

[0030] Figure 8A -B is a fluorescence imaging image of the in vitro internalization of the LY6A-bound small protein of the present invention.

[0031] Figure 9A -F is a graph showing the SPR curves of small protein conjugates purified at different concentrations.

[0032] Figure 10A -E shows a sagittal section of tissue from a mouse injected with the small protein of the present invention. Detailed Implementation

[0033] The present invention provides compositions and methods comprising small proteins as shuttles, which induce transcytosis across target tissues, for example, via astrocyte-specific receptors, neuron-specific receptors, carbonic anhydrase IV (CA-IV), low-density lipoprotein receptor-associated protein 6 (LRP6), or lymphocyte antigen 6 complex locus A (LY6A).

[0034] Small protein The interchangeable terms “small protein,” “computationally designed conjugate,” and “small-size conjugate” used in this article refer to a group of different protein scaffolds characterized by their small size, high stability, and multifunctionality in drug-like action.

[0035] Advantageously, small proteins can contain stable tertiary structures, which, in addition to resistance to proteolysis, reduction, and denaturation, can promote specificity and potency. Furthermore, their small size makes them potentially suitable for synthesis, full characterization, and regulation as small molecule drugs rather than biological products. Their size also facilitates computational design and SAR (Small Invasive Therapy), as well as good tissue penetration and manufacturing flexibility.

[0036] Based on the biophysical properties that influence their pharmacological capabilities and biosynthetic production strategies, some small proteins can be classified into three general subcategories: cystine-enhanced, hydrophobic nuclei, and chemically stable.

[0037] The largest small proteins are hydrophobic nucleoproteins, whose structure and stability are driven by rigid secondary structural elements (α-helices and β-sheets) arranged around a hydrophobic core, and whose folding is driven by the solvent repulsion of the hydrophobic core.

[0038] Examples of hydrophobic nucleoproteins include avidin, adnectin, centyrin, nanofittin, avidin, and funomer.

[0039] Adnectin and centyrin are based on fibronectin type 3 (10Fn3) domains, which are similar to the antibody VH domains with protruding loops extending from quasi-β-barrel structures. Fynomer is generated by modifying the SH3 domain of Fyn kinase and is a major β-barrel with an exposed loop for binding. Affinities are simple triple-helix domains based on the Z domain of protein A binding antibodies. Nanofittin and affitin are generated by modifying various 7-kDa DNA-binding proteins of thermophilic archaea and are extremely thermostable. All four classes do not require chemical or oxidative binding; thus, they can be produced in large quantities in bacteria. Binding of hydrophobic nucleoproteins is primarily driven by a strategy similar to that of antibody Fv domains, involving randomized sequences within loops or helices linked to rigid superstructures.

[0040] Cystine-enhanced small proteins use disulfides to drive folding and provide rigidity, where their folding and stability depend on Cys-Cys disulfides.

[0041] Examples of cystine-enhanced small proteins include affinity multimers, kunit domains, and cystine-dense peptides (CDPs).

[0042] Cystine-dense peptides (CDPs) are represented by structurally diverse scaffolds with similar biophysical properties, including natural cell permeabilizers or blood-brain barrier permeabilizer peptides. Advantageously, the significant protease resistance provided by cystine knots can enable activity in erosive environments such as the gastrointestinal (GI) tract. Kunitz domains are similar to CDPs but contain a hydrophobic nucleus and are specifically designed for protease inhibition; hundreds of such proteins exist in nature, such as the popular transplant scaffold human APPI. Affinity multimers are based on the ring-rich A domain of human cell surface receptors and, unlike CDPs or Kunitz domains, require calcium ion coordination. Affinity multimers have been shown to be highly suitable for polymerization. Although cystine-enhanced small proteins are smaller than hydrophobic nucleus small proteins, the screening strategies for cystine-enhanced small proteins are similar to those for larger scaffolds. Binding is conferred by identifying surface-exposed regions for transplantation of known binding motifs or randomized sequences, or by screening for mutations in the whole protein (with cysteine ​​retention).

[0043] Chemically stable small proteins are most similar to small molecule drugs in both size and function. While possessing unique properties, chemically stable small proteins can be broadly considered miniature, rigid versions of the β-sheets (β-hairpins), α-helices (bound peptides), or loops (bicyclics) found at natural protein-protein interfaces. All of these rely on chemical cross-linking steps to stabilize peptide sequences that would otherwise be unstable or unstructured.

[0044] Examples of chemically stable small proteins include β-hairpins, stapled peptides, and bicyclic compounds.

[0045] Trans-receptor payload delivery The present invention discloses methods and delivery systems for delivering payloads (e.g., therapeutic agents) across tissue surfaces via receptors that mediate transcytosis (e.g., astrocyte-specific receptors, neuron-specific receptors, CA-IV, LRP6, or LY6A).

[0046] The method includes providing a small protein capable of interacting with astrocyte-specific receptors, neuron-specific receptors, CA-IV, LRP6, or LY6A. The small protein may be part of a delivery system, and the delivery system may contain a payload to be delivered to the nervous system. The method may further include administering the delivery system to a subject.

[0047] In some embodiments, the delivery system comprises nanoparticles, nanotubes, nanowires, dendritic macromolecules, liposomes, liposomes and aqueous liposomes, polymer vesicles and nonionic surfactant vesicles, foams, hydrogels, cubes, quantum dots, exosomes, macrophages, and combinations thereof. In some embodiments, the delivery system comprises nanoparticles selected from: lipid-based nanoparticles, polymer 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.

[0048] For example, the payload may include antimicrobial agents, therapeutic agents, prodrugs, peptides, proteins, enzymes, lipids, biological response modulators, pharmaceuticals, lymphokines, heterologous antibodies or fragments thereof, detectable markers, polyethylene glycol (PEG) molecules, or combinations of two or more pharmaceuticals.

[0049] The payload may include neuroactive peptides, such as neurotrophic factors, endocrine factors, growth factors, paracrine factors, hypothalamic releasing factors, neurotransmitter peptides, peptide agonists of receptors expressed by CNS cells, peptides involved in lysosomal storage diseases, or any combination thereof. In another example, the payload may include IL-1 receptor antagonists (IL-1Ra), dalargin, interferon-β, glial-derived neurotrophic factor (GDNF), tumor necrosis factor receptor (TNFR), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophic factor-4 / 5, neurotrophic factor (NT)-3, neuro-rank protein, neuroregulatory protein, spindle protein, ciliary neurotrophic factor (CNTF), stem cell factor (SCF), brachiophyte protein, hepatocyte growth factor (HGF), and epidermal growth factor. Factors (EGF), transforming growth factor (TGF)-cx, TGF-β, vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), modulin, leukosheath embryonic factor, persephrine, interleukin, granulocyte colony-stimulating factor (CSF), granulocyte-macrophage-CSF, myocardial nutrient-1, hedgehog factor, leukemia inhibitory factor (LIF), metaphase factor, pleiotropic growth factor, erythropoietin (EPO), bone morphogenetic protein (BMP), spindle protein, sphingolipid activator protein, any fragment thereof, or any combination thereof.

[0050] Various aspects of the invention also provide for delivering the conjugate to a subject to transport the therapeutic agent across the BBB. In various aspects of the invention, the delivery of the therapeutic payload can be used to treat diseases, conditions, or lesions of the CNS. In various aspects of the invention, the therapeutic agent can be released from the conjugate after entering the CNS. In some respects, diseases, conditions, or lesions of the CNS can be, but are not limited to, 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 myelinolysis (CPM), adrenoleukodystrophy, Alexander's disease, Pelizaeus Merzbacher disease (PMZ), spherocytic leukoencephalopathy (Krabbe's disease), and Wallerian degeneration. Degeneration, optic neuritis, transverse myelitis, radiation injury, neurological complications of chemotherapy, acute ischemic optic neuropathy, vitamin E deficiency, isolated vitamin E deficiency syndrome, Bassen-Kornzweig syndrome, Marchifava-Bignami syndrome, metachromatic leukodystrophy, trigeminal neuralgia, Bell's palsy, primary tumor, secondary metastasis, or any combination thereof.

[0051] Carbonic anhydrase IV Carbonic anhydrase (or carbonic acid dehydrating enzyme) (CA) is a family of enzymes that catalyze the interconversion between carbon dioxide and water, as well as the dissociation of carbonic acid into ions. 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.

[0052] Carbonic anhydrase IV is an isoenzyme belonging to the carbonic anhydrase family, a zinc metalloenzyme family. This isoenzyme catalyzes the reversible reaction of CO2 hydration (H2O + CO2). Carbonic anhydrases (HCO3- + H+) enable enzymes to regulate the concentrations of CO2, H+, and HCO3- both intracellularly and extracellularly. They 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 anhydrases exhibit extensive diversity in tissue distribution and subcellular localization. Mammalian carbonic anhydrases have at least seven genetically distinct isoenzymes, designated I-VII, each of which catalyzes the reversible hydration of carbon dioxide via the zinc hydroxide mechanism. Physiological functions regulated by carbonic anhydrases include, for example, the removal of HCO3- from the lungs via respiration, the reuse of HCO3- in the kidneys, the production of aqueous humor in the eyes, the production of cerebrospinal fluid in the brain, the production of gastric juice in the stomach, pancreatic juice, and bone resorption by osteoclasts. Members of the carbonic anhydrase family also play important roles in metabolic processes including urea production, gluconeogenesis, and lipogenesis.

[0053] Unlike other soluble carbonic anhydrases or those attached to the plasma membrane via transmembrane domains, carbonic anhydrase IV is a glycosylphosphatidyl-inositol-anchored membrane isoenzyme. Carbonic anhydrase IV is widely conserved in vertebrates and exhibits a similar CNS expression profile in humans, with recent single-cell analysis of the human brain vascular system confirming its expression in the human brain's blood-brain barrier (BBB). Carbonic anhydrase IV has been shown to regulate pH, which is associated with neural firing, and can influence neuronal function through ion-gated channels.

[0054] In some embodiments, the carbonic anhydrase IV disclosed herein is human carbonic anhydrase IV. CA-IV is known to be located on the luminal surface of cerebral endothelial cells throughout the cortex and cerebellum, where it enzymatically regulates the carbon dioxide-bicarbonate balance. Human CA-IV has previously been characterized as a 35-kDa protein with “high activity” in CO2 hydration and higher activity than other isoenzymes in catalyzing HCO3- dehydration. Generally, human CA-IV contains an 18-amino acid signal sequence at the N-terminus of the protein translocated in the endoplasmic reticulum (ER), and a 260-amino acid “CA domain” containing the active site amino acid residues, showing 30-36% homology to cytoplasmic CA. At the C-terminus, an additional 27 amino acid residues, containing a hydrophobic sequence of 21 amino acids sufficient to cross the membrane, are preceded by a 6-amino acid signal sequence anchored to a GPI. Amino acid residue Ser 266 has been identified as the GPI anchor attachment site. Removing the C-terminal hydrophobic domain present in the CA-IV precursor has significant implications for GPI anchoring, cell surface expression, and enzyme activity. Based on the amino acid sequence deduced from the nucleotide sequence, human CA-IV does not contain the classic shared site (Asn-Xxx-Ser / Thr) for N-glycosylation. Human CA-IV also does not contain oligosaccharide chains, while other mammalian carbonic anhydrase IVs (e.g., mouse carbonic anhydrase IV) are glycoproteins with one or more oligosaccharide side chains.

[0055] In some embodiments, the carbonic anhydrase IV disclosed herein is mouse carbonic anhydrase IV. CA-IV has recently been found to be one of the mouse proteins most strongly positively correlated with plasma-protein uptake in the brain (slightly stronger than the commonly targeted transferrin receptor). This property can be used to identify receptors for enhanced BBB crossing. CA-IV is expressed in GI tracts that sense carbonation, kidneys and lungs, and taste receptor cells. Mouse and human CA-IV are highly homologous, containing the same amino acid at a position crucial to enzyme activity (e.g., histidine residue 64 (His 64)). Several differences include, for example, that mouse CA-IV is an N-linked glycoprotein and that the rate of CO2 hydration catalyzed by mouse CA-IV is much lower than that of human CA-IV. Without being bound by any theory, the lower enzymatic activity of mouse CA-IV may be related to several other amino acid substitutions, such as the substitution of Gly63 for Gln 63 in human CA-IV. Another difference between mouse and human CA-IV is the Val-131-Asp-136 segment (the segment of 130), which forms an α-helix in mice and an extended loop in human CA-IV.

[0056] In some embodiments, the carbonic anhydrase IV disclosed herein as a receptor for enhancing BBB crossing can be any carbonic anhydrase IV, such as mouse CA-IV, human CA-IV, or its homologs or variants. Carbonic anhydrase IV homologs and / or variants can be derived 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 publicly available databases identifiable by those skilled in the art, including, for example, UniProt, NCBI, and SwissProt.

[0057] In some embodiments, the small protein may interact with carbonic anhydrase IV (e.g., mouse CA-IV, human CA-IV, or homologs or variants thereof) disclosed herein, thereby increasing the permeability of the BBB (e.g., via transcytosis). In some embodiments, the increase in BBB permeability is achieved by altering (e.g., increasing or decreasing) the activity of carbonic anhydrase IV, such as by reducing its activity.

[0058] In some embodiments, the alteration of carbonic anhydrase IV activity is achieved through the interaction of a shuttle 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 may interact with the zinc binding site, the hydrophobic substrate binding pocket, or both.

[0059] The zinc binding site in carbonic anhydrase IV has a conserved structure dominated by a β-sheet superstructure, in which the metal-binding site is formed by at least three His residues. Without being bound by any theory, the zinc binding site is believed to be located on one face of the β-sheet at the base of a 15-Å deep conical active site fissure, where zinc is coordinated via three His residues and a hydroxide ion with a tetrahedral geometry. 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. Based on phylogenetic comparisons, this pocket is highly conserved across all active isoenzymes. Without being bound by any theory, the hydrophobic pocket is believed to have the minimum width and depth for efficient catalysis, and linear free energy relationships indicate that the volume of the amino acid residues at the base of the pocket and the hydrophobicity of the residues at the neck of the pocket are crucial to activity. Both the zinc binding site and the hydrophobic substrate-binding pocket in the carbonic anhydrase isoenzyme are highly conserved.

[0060] Low-density lipoprotein receptor-associated 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 containing LDLR-binding repeat sequences and EGF repeat sequences, and a related spacer subdomain containing a YWTD motif; a single transmembrane domain; and a C-terminal cytoplasmic domain, typically containing at least one copy of an NPXY motif.

[0061] Lrp6 is a single-pass transmembrane protein involved in the activation of the Wnt signaling pathway. Human LRP6 has 23 exons, is located on chromosome 12p13.2, and contains 1613 amino acids. LRP6 consists of four YWTD β-propeller domains, each followed by an EGF-like domain, three LDLR type A repeats, a transmembrane domain, and a short intracellular domain. It is structurally related to LRP5 and shares almost 71% homology at the nucleotide level. Approximately 85% of the 1613 amino acid length of Lrp6 is extracellular. Most extracellular ligands bind to LRP6 at the β-propeller. Each protein has a single-pass 22-amino acid segment across the cell membrane and a 207-amino acid segment located intracellularly.

[0062] Lymphocyte antigen 6 complex locus A (LY6A) Sca-1 is one of 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 the LU domain. The LU domain is a 60-80 amino acid domain composed of 6-10 cysteine ​​residues arranged in a specific spacing pattern that allows for the generation of different disulfide bridges of the three-fingered (3F) motif.

[0063] Although the direct homolog of Sca-1 is absent in humans, human chromosome 8 (the syntenic region of mouse chromosome 15) carries several genes containing the characteristic LU domain. The LU domain is present in the extracellular domains of cell surface receptors with transmembrane domains (activin type 2 receptor and bone morphogenetic IA receptor), or in the GPI-anchored protein CD177 or secreted globular proteins such as the CD59 antigen SLURP1 / 2.

[0064] Experimental Examples Small protein conjugates are computationally designed and customized for specific receptors, intended not only for crossing the blood-brain barrier (BBB) ​​but also for receptor-mediated delivery to other organs. The proprietary design methodology behind these small proteins ensures they possess epitope information, providing precise binding to the targeted receptor. These designed small proteins enable specific and efficient therapeutic delivery.

[0065] Approximately 200 small protein conjugates targeting different BBB receptors (such as mouse LY6A, human CA-IV, and human LRP6) were designed and screened for their targets. Surface plasmon resonance (SPR) was used to demonstrate that the small proteins of this invention bind to their target receptors. The small proteins were shown to be further internalized in cells overexpressing the target receptors. To demonstrate the in vivo application potential of the small protein conjugates, the in vivo distribution of small proteins targeting BBB receptors found in rodent brains was characterized.

[0066] Figure 1 This invention describes a three-stage workflow for the design and screening of small proteins.

[0067] In Phase 1, receptor targets are analyzed to develop a design strategy: 1a) Binding motif identification: Identification of experimentally validated receptor binding motifs through literature or in vitro drop-down selection; 1b) Binding structure analysis: Use protein databases (PDB) or prediction tools such as AlphaFold-Multimer to obtain the complex structures of binding motifs and receptors; 1c) Design strategy formation: Based on existing information, a design strategy, such as epitope targeting or motif scaffolding, was determined.

[0068] In phase 2, computer-simulated small protein sequences were designed and evaluated: 2a) Main chain generation: Use diffusion-based generative AI (e.g., Rfdiffusion) to generate the main chain structure model; 2b) Sequence design: Use tools such as ProteinMPNN to reverse fold the structural model to generate protein sequences that may fold into the desired structure; 2c) Design evaluation: The designed protein sequences were evaluated using AlphaFold confidence scores and APPRAISE rankings.

[0069] In the third stage, the designed sequences are screened and characterized: 3a) Cell-free protein synthesis: using a cell-free protein production system to synthesize small amounts of protein; 3b) Use surface plasmon resonance (SPR) or cell-based binding assays to screen for the generated proteins; 3c) Single protein characterization: Hits from screening were expressed in bacteria and their in vitro and in vivo binding, endocytosis and transcytosis properties were subsequently evaluated.

[0070] Cell-free design of small proteins Following the manufacturer's protocol, two batches of the designed small protein were fused with HA tags and synthesized using NEBPureExpress, a cell-free protein production system based on E. coli. Western blotting was used to examine expression in 1 μL of each reaction.

[0071] Figure 2A -B is an image of the protein blot of the synthesis of the small protein of the present invention.

[0072] The blots were stained with 1:1000 anti-HA-HRP and the bands were developed using UltraTMB substrate. The results showed that most of the designed small proteins could be successfully produced at concentrations of 0.5–5 μmol.

[0073] Small protein screening 200 nM Fc-labeled LY6A receptor protein was diluted in HBS-EF+ buffer containing 1% BSA and immobilized on a protein A chip. Small proteins were synthesized using a cell-free reaction, diluted in the same buffer as the receptor protein, and then flow-through onto the chip.

[0074] Figure 3A -P is a graph of the SPR curve of LY6A binding to small proteins in this invention.

[0075] The designed small proteins, especially those marked with circles (XMP-6, XMP-7, XMP-34), show binding signals. Note that the y-axis range and sample concentration may differ between the different plots.

[0076] 200 nM Fc-labeled human CA-IV receptor protein was diluted in HBS-EF+ buffer containing 1% BSA and immobilized on a protein A chip. Small proteins were synthesized using a cell-free reaction, diluted in the same buffer as the receptor protein, and then flow-through onto the chip.

[0077] Figure 4AA-4BT This is a graph showing the SPR curve of the CA-IV binding small protein of the present invention.

[0078] The designed small proteins, especially those with circles (XMP-27 and XMP-52), show binding signals. Note that the y-axis range and sample concentration may differ between the different plots.

[0079] 200 nM Fc-labeled human LRP6 receptor protein was diluted in HBS-EF+ buffer containing 1% BSA and immobilized on a protein A chip. Small proteins were synthesized using a cell-free reaction, diluted in the same buffer as the receptor protein, and then flow-through onto the chip.

[0080] Figure 5A -5J is a graph showing the SPR curve of LRP6 binding to small proteins in this invention.

[0081] The designed small proteins, especially those with circles (XMP-42 and XMP-41), show binding signals. Note that the y-axis range and sample concentration may differ between the different plots.

[0082] In vitro assay The in vitro internalization of the designed LY6A-binding small protein was analyzed. Cultured HEK293 cells were transfected with receptor DNA and incubated with 0.1 μmol of HA-labeled small protein for 1 hour, followed by fixation with 4% PFA and optionally infiltration with 0.3% Triton-X100. Cells were then stained and imaged with a fluorescently labeled anti-HA primary antibody.

[0083] Figure 6A -D is a fluorescence imaging image of the in vitro internalization of the LY6A-bound small protein of the present invention.

[0084] like Figure 6A As shown in the image, HEK293 cells incubated with the small protein XMP-6 exhibit specific enrichment of XMP-6 in cells overexpressing the LY6A receptor.

[0085] like Figure 6B As shown, Z-sections of representative cells reveal internal particle signals that may indicate endocytosis.

[0086] like Figure 6C As shown, images of HEK293 cells incubated with small proteins XMP-7, XMP-8, and XMP-34 reveal that the enriched signal is not present in all the designed small proteins, including those that share the same binding motif as XMP-6 but have different scaffolds (e.g., XMP-7 and XMP-8).

[0087] The in vitro internalization of the designed CA-IV-binding small protein was analyzed. Cultured HEK293 cells were transfected with receptor DNA and incubated with 0.1 μmol of HA-labeled small protein for 1 hour, followed by fixation with 4% PFA and optionally infiltration with 0.3% Triton-X100. Cells were then stained and imaged with a fluorescently labeled anti-HA primary antibody.

[0088] Figure 7A -B is a fluorescence imaging image of the in vitro internalization of the CA-IV-binding small protein of the present invention.

[0089] The images show HEK293 cells incubated with the small proteins XMP-27, XMP-28, XMP-39, and XMP-52. Intracellular fluorescence signals are enhanced only in permeable cells overexpressing the CA-IV receptor, suggesting potential internalization of the small proteins.

[0090] The in vitro internalization of the designed LRP6 binding small proteins XMP-184, XMP-185 and XMP-198 was analyzed.

[0091] The HA-labeled small protein was incubated with HeLa cells expressing the human LRP6 E1 / E2 domain at 37°C for 1 hour at 2.5 µM. The supernatant and purified fractions were then immunostained.

[0092] Figure 8A -B is a fluorescence imaging image of the in vitro internalization of the LY6A-bound small protein of the present invention.

[0093] The selected small protein was expressed in *E. coli* and purified using Ni-NTA-based single-step affinity purification. SPR assays were used to assess the binding interaction between the receptor protein and the purified small protein. 200 nM Fc-labeled receptor protein was immobilized on a protein A-pre-coated capture sensor, and then different concentrations of engineered small protein were introduced.

[0094] Figure 9A -F is a graph showing the SPR curves of small protein conjugates purified at different concentrations.

[0095] The receptor concentration is indicated in the illustration.

[0096] In vivo assay The designed mouse BBB receptor-binding small protein was characterized in vivo. C57BL / 6J mice (LY6A+) and BALB / cJ mice (LY6a-) were injected with 0.05 mg of HA-labeled small protein via retroorbital injection, and tissues were collected at specified time points, then fixed and stained with HA labeling.

[0097] Figure 10A -E shows a sagittal section of tissue from a mouse injected with the small protein of the present invention.

[0098] like Figure 10A As shown, images of sagittal sections reveal LY6A-dependent XMP-6 enrichment across the brains of C57BL / 6J mice (LY6A+), but not in the brains of BALB / cJ mice (LY6a-).

[0099] like Figure 10B As shown in the image, the liver images reveal that XMP-6 is present in the liver regardless of LY6A expression.

[0100] like Figure 10C As shown in Figure D, magnified brain slice images indicate that XMP-6 is enriched near cerebral blood vessels.

[0101] like Figure 10E As shown, a brain slice image of XMP-28 (a promising mouse CA-IV binding small protein) shows that it is enriched near cerebral blood vessels.

[0102] in conclusion These small proteins bind efficiently to the BBB receptor and accumulate in the brains of mice expressing LY6A upon systemic injection. These receptor-binding small proteins have shown promise as a powerful tool for delivering therapeutics and diagnostics to specific organs.

[0103] By incorporating references This disclosure references and cites other documents, such as patents, patent applications, patent publications, magazines, books, papers, and web content. All such documents are hereby incorporated in their entirety by reference for all purposes.

[0104] equivalent Based on the entire contents of this document, including references to scientific and patent literature cited herein, various modifications to the invention and many other embodiments thereof will become apparent to those skilled in the art, except for those shown and described herein. The subject matter of this document contains important information, illustrations, and guidance that can be adapted to practice the invention in its various embodiments and equivalents.

Claims

1. A conjugate comprising: A shuttle, comprising a small protein that binds to a portion of a cell receptor mediating transcytosis; and Therapeutic cargo, which is attached to the shuttle.

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

3. The conjugate of claim 1, wherein the small protein binds to the cell receptor mediating transcytosis, the small protein comprising a sequence having at least 95% sequence content of a sequence selected from SEQ ID NO: 1-248.

4. The conjugate of claim 1, wherein the small protein binds to the cell receptor LY6A that mediates transcytosis, and comprises a sequence having at least 95% sequence content of a sequence selected from the following: 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 small protein binds to the CA-IV cell receptor mediating transcytosis, and comprises a sequence having at least 95% sequence content of a sequence selected from the following: 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 small protein binds to the cell receptor LRP6 that mediates transcytosis, and comprises a sequence having at least 95% sequence content of a sequence selected from the following: 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 small protein binds to an astrocyte-specific or neuron-specific receptor and comprises a sequence having at least 95% sequence content of a sequence selected from SEQ ID NO: 50-51 or SEQ ID NO:

127.

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

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 biomolecule.

11. The conjugate according to claim 10, wherein the biomolecule 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 small protein is in monomeric form, bispecific form, multispecific form, or as part of an alternative protein scaffold.

13. The conjugate according to claim 1, wherein the conjugate is characterized in that it delivers 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 treating a condition affecting the central nervous system.

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

16. A method for delivering therapeutic goods across a subject's tissues, the method comprising: The subject is provided with a conjugate comprising: A shuttle, comprising a small protein that binds to a portion of a cell receptor mediating transcytosis; and Therapeutic cargo, which is attached to the shuttle.

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

18. The method of claim 1, wherein the small protein binds to the cell receptor mediating transcytosis, the small protein comprising a sequence having at least 95% sequence content of a sequence selected from SEQ ID NO: 1-248.

19. The method of claim 1, wherein the small protein binds to the cell receptor LY6A that mediates transcytosis, and comprises a sequence having at least 95% sequence content of a sequence selected from the following: 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 small protein binds to the cell receptor CA-IV that mediates transcytosis, and comprises a sequence having at least 95% sequence content of a sequence selected from the following: 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 small protein binds to the cell receptor LRP6 that mediates transcytosis, and comprises a sequence having at least 95% sequence content of a sequence selected from the following: 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 small protein binds to an astrocyte-specific or neuron-specific receptor and comprises a sequence having at least 95% sequence content of 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 attached to the shuttle via a connector.

24. The method of claim 1, wherein the therapeutic cargo is covalently coupled to the shuttle.

25. The method of claim 1, wherein the therapeutic cargo is a biomolecule.

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

27. The method of claim 1, wherein the small protein is in monomeric form, bispecific form, multispecific form, or as part of an alternative protein scaffold.

28. The method of claim 1, wherein the conjugate is characterized in that it delivers the therapeutic cargo across the blood-brain barrier (BBB).

29. The method of claim 13, wherein the therapeutic cargo is a therapeutic cargo for treating a condition affecting the central nervous system.

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

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