Compositions and methods for internalizing enzymes

Patent metadata

Field	Value
Publication number	US-2018355017-A1
Application number	US-201816001717-A
Country	US
Kind code	A1
Filing date	Jun 6, 2018
Priority date	Jun 7, 2017
Publication date	Dec 13, 2018
Grant date	—

Abstract

Compositions and methods for treating enzyme-deficiency diseases are disclosed. Multidomain therapeutic proteins containing an internalization effector binding domain and a lysosomal replacement enzyme activity are disclosed. The multidomain therapeutic proteins are capable of entering cells, segregating to the lysosome, and delivering the replacement enzyme activity to the lysosome.

First claim

What is claimed is:

1 . A polynucleotide encoding a multidomain therapeutic protein comprising a delivery domain and an enzyme domain, wherein the delivery domain binds to an internalization effector.

2 . The polynucleotide of claim 1 , wherein the delivery domain binds to an internalization effector selected from the group consisting of CD63, Integrin alpha-7 (ITGA7), MHC-I, Kremen-1, Kremen-2, LRP5, LRP6, LRP8, transferrin receptor, LDL-receptor, LDL-related protein 1 receptor, ASGR1, ASGR2, amyloid precursor protein-like protein-2 (APLP2), apelin receptor (APLNR), MAL (myelin and lymphocyte protein (MAL), IGF2R, vacuolar-type H+ ATPase, diphtheria toxin receptor, folate receptor, glutamate receptors, glutathione receptor, leptin receptors, scavenger receptor A1-5 (SCARA1-5), SCARB1-3, and CD36.

3 . The polynucleotide of claim 2 , wherein the delivery domain binds to the internalization effector CD63.

4 . The polynucleotide of claim 3 , wherein the delivery domain comprises
an amino acid sequence of SEQ ID NO:2, or an amino acid sequence selected from the group consisting of an amino acid sequence of an HCVR set forth in Table 11, an amino acid sequence of a LCVR set forth in Table 11, an amino acid sequence of an HCDR1 set forth in Table 11, an amino acid sequence of an HCDR2 set forth in Table 11, an amino acid sequence of an HCDR3 set forth in Table 11, an amino acid sequence of a LCDR1 set forth in Table 11, an amino acid sequence of a LCDR2 set forth in Table 11, an amino acid sequence of a LCDR3 set forth in Table 11, an HCVR/LCVR amino acid pair set forth as SEQ ID NOs: 14/22, SEQ ID NOs: 30/38, SEQ ID NOs: 46/54, or SEQ ID NOs: 62/70, wherein the HCVR/LCVR amino acid pair comprises from N-terminal to C-terminal: the HCVR, an optional linker, and the LCVR.

5 . The polynucleotide of claim 1 , wherein the enzyme domain comprises a hydrolase.

6 . The polynucleotide of claim 1 , wherein the enzyme domain comprises a glycosylase.

7 . The polynucleotide of claim 1 , wherein the enzyme domain comprises a glycosidase.

8 . The polynucleotide of claim 1 , wherein the enzyme domain comprises an alpha-glucosidase.

9 . The polynucleotide of claim 1 , wherein the enzyme domain comprises an amino acid sequence of SEQ ID NO:1, SEQ ID NO:13, SEQ ID NO:78, or a fragment thereof.

10 . The polynucleotide of claim 1 further comprising a virus nucleic acid sequence and/or a locus-targeting nucleic acid sequence.

11 . The polynucleotide of claim 1 , further comprising a virus nucleic acid sequence and at least one of (i) a locus-targeting nucleic acid sequence, (ii) a tissue specific regulatory element, or (iii) a combination of (i) and (ii).
wherein the virus nucleic acid sequence is an adeno-associated virus (AAV) nucleic acid internal terminal repeat sequence comprising a sequence set forth as SEQ ID NO:6, SEQ ID NO:7, or a combination thereof, wherein the locus-targeting nucleic acid sequence targets the polynucleotide to a locus is selected from the group consisting of an EESYR locus, a SARS locus, position 188,083,272 of human chromosome 1 or its non-human mammalian orthologue, position 3,046,320 of human chromosome 10 or its non-human mammalian orthologue, position 67,328,980 of human chromosome 17 or its non-human mammalian orthologue, an adeno-associated virus site 1 (AAVS1) on chromosome, a naturally occurring site of integration of AAV virus on human chromosome 19 or its non-human mammalian orthologue, a chemokine receptor 5 (CCR5) gene, a chemokine receptor gene encoding an HIV-1 coreceptor, a mouse Rosa26 locus or its non-murine mammalian orthologue, and a human albumin (alb) locus, and wherein the tissue specific regulatory element is a liver specific enhancer comprising the sequence set forth as SEQ ID NO: 9 and/or a liver specific promoter comprising the sequence set forth as SEQ ID NO:10.

12 . The polynucleotide of claim 1 , wherein the polynucleotide comprises a nucleic acid sequence of SEQ ID NO:11 and/or encodes an amino acid sequence set forth as SEQ ID NO:10 or SEQ ID NO:78.

13 . A composition comprising a gene therapy vector comprising a polynucleotide of claim 1 ,
wherein the gene therapy vector is selected from the group consisting of (i) a viral vector comprising the polynucleotide, (ii) the polynucleotide by itself as a naked polynucleotide, (iii) a complex comprising the polynucleotide complexed with a metal, a cation, an anion, a lipid, a polymer, and any combination thereof, and (iv) any combination of (i)-(iii).

14 . The composition of claim 13 , 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.

15 . The composition of claim 14 , wherein the gene therapy vector is an AAV2/8 chimera.

16 . The composition of claim 13 , further comprising a pharmaceutically acceptable carrier, wherein the composition is a pharmaceutically acceptable composition for administration into a subject, and
wherein the composition comprises a therapeutically effective amount of the polynucleotide.

17 . A recombinant multidomain therapeutic protein comprising a delivery domain and an enzyme domain, wherein the protein is encoded by the polynucleotide according to claim 1 .

18 . The recombination multidomain therapeutic protein of claim 17 , comprising the sequence set forth as SEQ ID NO:10 or SEQ ID NO:79.

19 . A method of expressing in a cell a recombinant multidomain therapeutic protein comprising a delivery domain and an enzyme domain, the method comprising:
a. contacting the cell with the composition of claim 13 ; b. allowing the polynucleotide to integrate into a genomic locus of the cell; and c. allowing the cell to produce the recombinant multidomain therapeutic protein.

20 . The method of claim 19 , wherein the genomic locus of the cell is a safe harbor locus selected from the group consisting of an EESYR locus, a SARS locus, position 188,083,272 of human chromosome 1 or its non-human mammalian orthologue, position 3,046,320 of human chromosome 10 or its non-human mammalian orthologue, position 67,328,980 of human chromosome 17 or its non-human mammalian orthologue, an adeno-associated virus site 1 (AAVS1) on chromosome, a naturally occurring site of integration of AAV virus on human chromosome 19 or its non-human mammalian orthologue, a chemokine receptor 5 (CCR5) gene, a chemokine receptor gene encoding an HIV-1 coreceptor, a mouse Rosa26 locus or its non-murine mammalian orthologue, and a human albumin (alb) locus.

21 . The method of claim 19 , wherein the polynucleotide comprises a nucleic acid sequence of SEQ ID NO:11 and/or encodes an amino acid sequence set forth as SEQ ID NO:10 or SEQ ID NO:79.

22 . A method of reducing glycogen accumulation in a tissue in a patient in need thereof, treating enzyme deficiency in a patient in need thereof, and/or tolerizing the patient to the enzyme for which it is deficient comprising administering to the patient the composition of claim 16 , wherein the patient is deficient in GAA, and wherein the enzyme domain comprises GAA or a portion thereof.

23 . The method of claim 22 , wherein the tissue is selected from the group consisting of heart, liver, and skeletal muscle.

24 . The method of claim 22 , wherein high serum levels of GAA are maintained for at least 12 weeks.

25 . The

Assignees

• Regeneron Pharma

Inventors

• Baik Andrew
• Cygnar Katherine
• Schoenherr Christopher
• Kyratsous Christos
• Wang Cheng

Classifications

• C07K2317/77

  Internalization into the cell · CPC title

• C07K16/2896 Primary

  against molecules with a "CD"-designation, not provided for elsewhere · CPC title

• C12N9/2402

  hydrolysing O- and S- glycosyl compounds (3.2.1) · CPC title

• A61P43/00

  Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 · CPC title

• C07K2317/24

  containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered · CPC title