# US 2020/0399623 A1 · Methods and compositions for therapeutic protein delivery > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2020/0399623 A1 - Document ID: US20200399623A1 - Status: Published application - Kind: A1 - Application number: 16/968,452 - Filed: 2019-02-07 - Published: 2020-12-24 - Earliest claimed: 2018-02-07 - Assignees: Regeneron Pharmaceuticals, Inc. - Applicants: Regeneron Pharmaceuticals, Inc. - Inventors: Andrew Baik; Katherine Cygnar; Maria Praggastis - Web page: https://patentgrep.com/patent/US20200399623A1 ## CPC - A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 - C12N 9/2408: {acting on alpha -1,4-glucosidic bonds} - A61K 38/47: acting on glycosyl compounds (3.2), e.g. cellulases, lactases - A61K 47/6815: {Enzymes} - A61K 47/6849: {the antibody targeting a receptor, a cell surface antigen or a cell surface determinant} - A61K 47/6879: {the immunoglobulin having two or more different antigen-binding sites, e.g. bispecific or multispecific immunoglobulin} - A61K 47/6881: {Cluster-antibody conjugates, i.e. the modifying agent consists of a plurality of antibodies covalently linked to each other or of different antigen-binding fragments covalently linked to each other} - A61K 47/6901: {Conjugates being cells, cell fragments, viruses, ghosts, red blood cells or viral vectors} - A61K 48/005: {characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered} - A61P 3/00: Drugs for disorders of the metabolism (of the blood or the extracellular fluid A61P7/00) - C07K 16/2839: {against the integrin superfamily} - C07K 16/2881: {against CD71} - C07K 16/2896: {against molecules with a "CD"-designation, not provided for elsewhere} - C12Y 302/0102: Alpha-glucosidase (3.2.1.20) - A01K 2207/15: Humanized animals - A01K 2217/072: maintaining or altering function, i.e. knock in - A01K 2217/075: inducing loss of function, i.e. knock out - A01K 2217/15: Animals comprising multiple alterations of the genome, by transgenesis or homologous recombination, e.g. obtained by cross-breeding - A01K 2227/105: Murine - A01K 2267/0306: Animal model for genetic diseases - A61K 2039/505: {comprising antibodies} - C07K 2317/622: Single chain antibody (scFv) - C07K 2319/33: fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies - C12N 2750/14143: viral genome or elements thereof as genetic vector - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - C12Y: ENZYMES - A01K: ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS ## Abstract Compositions and methods for delivering a therapeutic protein to the central nervous system (CNS), in order to treat diseases and disorders that impair the CNS, such as treating lysosomal storage diseases are disclosed. Therapeutic proteins delivered via a therapeutically effective amount of a nucleotide composition encoding the therapeutic protein conjugated to a cell surface receptor-binding protein that crosses the blood brain barrier (BBB) are provided. ## Claims ### Claim 1 A method of delivering a therapeutic protein to the central nervous system (CNS) of a patient, comprising delivering a nucleotide composition encoding a multidomain therapeutic protein to the liver in the patient to form a liver depot for the production and secretion of the multidomain therapeutic at a consistent serum level of at least 1 μg/mL over consecutive days, weeks or months following delivery to provide a therapeutically effective amount of the multidomain therapeutic protein in the CNS, wherein the multidomain therapeutic protein comprises an antibody or antigen-binding portion thereof that binds CD63 or ITGA7 and an enzyme domain. ### Claim 2 (depends on 1) The method of claim 1, wherein the therapeutic protein is a lysosomal enzyme. ### Claim 3 (depends on 2) The method of claim 2, wherein the lysosomal enzyme is GAA. ### Claim 4 (depends on 1) The method of claim 1, wherein the nucleotide composition is administered via a viral vector. ### Claim 5 (depends on 4) The method of claim 4, wherein the viral vector is an AAV vector, optionally wherein the nucleotide composition is administered at a dose of at least 2×1012 viral genomes per kilogram (vg/kg). ### Claim 6 (depends on 1) The method of claim 1, wherein the antibody or antigen-binding portion thereof comprises a single-chain variable fragment (scFv). ### Claim 7 (depends on 1) The method of claim 1, wherein the antibody or antigen-binding portion thereof comprises an amino acid sequence of SEQ ID NO:2. ### Claim 8 (depends on 1) The method of claim 1, wherein the therapeutic protein comprises a hydrolase. ### Claim 9 (depends on 1) The method of claim 1, wherein the therapeutic protein comprises a glycosylase. ### Claim 10 (depends on 1) The method of claim 1, wherein the therapeutic protein comprises a glycosidase. ### Claim 11 (depends on 1) The method of claim 1, wherein the therapeutic protein comprises an alpha-glucosidase. ### Claim 12 (depends on 1) The method of claim 1, wherein the therapeutic protein comprises an amino acid sequence of SEQ ID NO:1 or SEQ ID NO:13, or a fragment thereof. ### Claim 13 (depends on 1) The method of claim 1, wherein the nucleotide composition comprises a nucleic acid sequence of SEQ ID NO:11. ### Claim 14 (depends on 1) The method of claim 1, wherein the enzyme domain comprises an alpha-glucosidase, and wherein the glycogen level(s) in any CNS tissue in the subject is reduced for at least nine months post-treatment. ### Claim 15 (depends on 1) The method of claim 1, wherein the subject has Pompe disease. ### Claim 16 (depends on 1) The method of claim 1, wherein the serum level is at least 2 μg/mL. ### Claim 17 (depends on 1) The method of claim 1, wherein the antibody or antigen-binding portion thereof binds an extracellular domain of CD63 or ITGA7. ### Claim 18 A multidomain therapeutic protein comprising two or more delivery domains and an enzyme domain, wherein one of the two or more delivery domains binds human transferrin receptor (hTfR), and wherein an other of the two or more delivery domains comprises an antibody or antigen-binding portion thereof that binds CD63 or ITGA7. ### Claim 19 (depends on 18) The multidomain therapeutic protein of claim 18, wherein each of the two or more delivery domains comprises an antigen-binding protein. ### Claim 20 (depends on 18) The multidomain therapeutic protein of claim 18, wherein at least one of the two or more delivery domains comprises a single-chain variable fragment (scFv). ### Claim 21 (depends on 18) The multidomain therapeutic protein of claim 18, wherein at least one of the two or more delivery domains comprises a half-body. ### Claim 22 (depends on 21) The multidomain therapeutic protein of claim 21, wherein the delivery domain that binds hTfR is an scFv, wherein the half-body binds CD63, wherein the enzyme domain is GAA, and wherein GAA is conjugated to the carboxy terminus of the half-body that binds CD63. ### Claim 23 (depends on 22) The multidomain therapeutic protein of claim 22, wherein each of the two or more delivery domains comprises an scFv. ### Claim 24 (depends on 20) The multidomain therapeutic protein of claim 20, wherein at least one scFv is fused to an Fc. ### Claim 25 (depends on 24) The multidomain therapeutic protein of claim 24, wherein the Fc comprises a wildtype human IgG4 isotype, or derivative thereof. ### Claim 26 (depends on 24) The multidomain therapeutic protein of claim 24, wherein GAA is conjugated to the carboxy terminus of the Fc. ### Claim 27 (depends on 23) The multidomain therapeutic protein of claim 23, comprising an anti-hTfR scFv and an anti-hCD63 scFv. ### Claim 28 (depends on 27) The multidomain therapeutic protein of claim 27, wherein the anti-hTfR scFv and anti-hCD63 scFv are both linked, at their carboxy termini, to a single GAA enzyme. ### Claim 29 (depends on 18) The multidomain therapeutic protein of claim 18, wherein the delivery domain is an anti-hTfR scFv, and the enzyme domain is linked to carboxy terminus of the VL domain of the scFv. ### Claim 30 (depends on 29) The multidomain therapeutic protein of claim 29, comprising the other delivery domain linked to the N-terminus of the VH domain of the anti-hTfR scFv. ### Claim 31 (depends on 30) The multidomain therapeutic protein of claim 30, wherein the other delivery domain is an anti-hCD63 scFv. ### Claim 32 (depends on 18) The multidomain therapeutic protein of any one claim 18, wherein the enzyme domain comprises the amino acid sequence set forth as SEQ ID NO:1. ### Claim 33 A multidomain therapeutic protein comprising at least two delivery domains and at least one enzyme domain, wherein each of the at least two delivery domains is independently selected from the group consisting of an antibody, a half-body, and an scFv, and wherein one or more of the delivery domains is associated the at least one enzyme domain, preferably wherein the one or more delivery domains is covalently linked to the at least one enzyme domain. ### Claim 34 (depends on 33) The multidomain therapeutic protein of claim 33, comprising a structure as depicted in FIG. 1C, FIG. 1D, FIG. 1E, or FIG. 1F. ### Claim 35 (depends on 18) A polynucleotide encoding the multidomain therapeutic protein of claim 18. ### Claim 36 (depends on 35) The polynucleotide of claim 35, further comprising a virus nucleic acid sequence and a locus-targeting nucleic acid sequence. ### Claim 37 (depends on 35) The polynucleotide of claim 35, further comprising a virus nucleic acid sequence and a locus-targeting nucleic acid sequence, wherein the virus nucleic acid sequence is an adeno-associated virus (AAV) nucleic acid sequence. ### Claim 38 (depends on 35) The polynucleotide of claim 35, further comprising a virus nucleic acid sequence and a locus-targeting nucleic acid sequence, wherein the virus nucleic acid sequence is an adeno-associated virus (AAV) nucleic acid sequence, and wherein the AAV nucleic acid sequence comprises an internal terminal repeat sequence, and optionally, a tissue specific regulatory element such as a liver specific promoter or a neuronal specific promoter. ### Claim 39 (depends on 35) The polynucleotide of claim 35, further comprising a virus nucleic acid sequence and a locus-targeting nucleic acid sequence, wherein the virus nucleic acid sequence is an adeno-associated virus (AAV) nucleic acid sequence comprising an internal terminal repeat sequence that comprises SEQ ID NO:6, SEQ ID NO:7, or both, and optionally, a tissue specific regulatory element such as a liver specific promoter or a neuronal specific promoter. ### Claim 40 (depends on 35) The polynucleotide of claim 35, further comprising a tissue specific regulatory element comprising the sequence set forth as SEQ ID NO:8 and/or SEQ ID NO:9. ### Claim 41 (depends on 35) A gene therapy vector comprising a polynucleotide of claim 35. ### Claim 42 (depends on 41) The gene therapy vector of claim 41, wherein the gene therapy vector is selected from the group consisting of a viral vector, optionally wherein the viral vector is a natural virus, an engineered virus, or a chimeric virus, a naked polynucleotide comprising the polynucleotide of encoding a multidomain therapeutic protein comprising two or more delivery domains and an enzyme domain, wherein one of the two or more delivery domains binds human transferrin receptor (hTfR), and wherein an other of the two or more delivery domains comprises an antibody or antigen-binding portion thereof that binds CD63 or ITGA7, a polynucleotide complex, optionally wherein the polynucleotide complex is a lipid nanoparticle comprising the polynucleotide encoding a multidomain therapeutic protein comprising two or more delivery domains and an enzyme domain, wherein one of the two or more delivery domains binds human transferrin receptor (hTfR), and wherein an other of the two or more delivery domains comprises an antibody or antigen-binding portion thereof that binds CD63 or ITGA7 and lipids, and any combination thereof. ### Claim 43 (depends on 41) The gene therapy vector of claim 41, wherein the gene therapy vector is a viral vector selected from the group consisting of a retrovirus, adenovirus, herpes simplex virus, pox virus, vaccinia virus, lentivirus, or an adeno-associated virus. ### Claim 44 (depends on 42) The gene therapy vector of claim 42, wherein the gene therapy vector is AAV9, Anc80, an AAV2/8 chimera and/or an AAV pseudotyped to a specific tissue, e.g., the liver or neuronal tissue. ### Claim 45 (depends on 18) Use of a nucleotide that encodes the multidomain therapeutic protein of claim 18, in a method of delivering a therapeutic protein to the central nervous system (CNS) of a patient, the method comprising delivering the nucleotide composition encoding the multidomain therapeutic protein to the liver of the patient to form a liver depot for the production and secretion of the multidomain therapeutic at a consistent serum level of at least 1 μg/mL over consecutive days, weeks or months following delivery to provide a therapeutically effective amount of the multidomain therapeutic protein in the CNS, wherein the multidomain therapeutic protein comprises an antibody or antigen-binding portion thereof that binds CD63 or ITGA7 and an enzyme domain. ### Claim at position 46 46-65. (canceled) ### Claim 66 (depends on 35) Use of the polynucleotide of claim 35 in a method of delivering a therapeutic protein to the central nervous system (CNS) of a patient, comprising delivering the polynucleotide encoding the multidomain therapeutic protein to the liver of the patient to form a liver depot for the production and secretion of the multidomain therapeutic at a consistent serum level of at least 1 μg/mL over consecutive days, weeks or months following delivery to provide a therapeutically effective amount of the multidomain therapeutic protein in the CNS, wherein the multidomain therapeutic protein comprises an antibody or antigen-binding portion thereof that binds CD63 or ITGA7 and an enzyme domain. ### Claim 67 (depends on 41) Use of a nucleotide that encodes the gene therapy vector of claim 41 in a method of delivering a therapeutic protein to the central nervous system (CNS) of a patient, comprising delivering the gene therapy vector to the liver of the patient to form a liver depot for the production and secretion of the multidomain therapeutic at a consistent serum level of at least 1 μg/mL over consecutive days, weeks or months following delivery to provide a therapeutically effective amount of the multidomain therapeutic protein in the CNS, wherein the multidomain therapeutic protein comprises an antibody or antigen-binding portion thereof that binds CD63 or ITGA7 and an enzyme domain.