# US 12,215,138 B2 · Compositions and methods for internalizing enzymes

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- Number: US 12,215,138 B2
- Document ID: US12215138B2
- Status: Granted
- Kind: B2
- Application number: 17/528,828
- Filed: 2021-11-17
- Published: 2022-06-23
- Granted: 2025-02-04
- Earliest claimed: 2017-06-07
- Assignees: Regeneron Pharmaceuticals, Inc.
- Applicants: Regeneron Pharmaceuticals, Inc.
- Inventors: Andrew Baik; Katherine Cygnar; Christopher Schoenherr; Christos Kyratsous; Cheng Wang
- Web page: https://patentgrep.com/patent/US12215138B2

## CPC

- C07K 14/70596: {Molecules with a "CD"-designation not provided for elsewhere}
- A61K 38/47: acting on glycosyl compounds (3.2), e.g. cellulases, lactases
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C07K 16/2896: {against molecules with a "CD"-designation, not provided for elsewhere}
- C12N 9/14: Hydrolases (3)
- C12N 9/24: acting on glycosyl compounds (3.2)
- C12N 9/2402: {hydrolysing O- and S- glycosyl compounds (3.2.1)}
- C12N 9/2428: {Glucan 1,4-alpha-glucosidase (3.2.1.3), i.e. glucoamylase}
- A61K 48/00: Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- C07K 2317/24: containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
- C07K 2317/31: multispecific
- C07K 2317/622: Single chain antibody (scFv)
- C07K 2317/77: Internalization into the cell
- C07K 2317/92: Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
- C07K 2319/00: Fusion polypeptide
- C07K 2319/33: fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
- C12N 2710/10043: viral genome or elements thereof as genetic vector
- C12N 2750/14143: viral genome or elements thereof as genetic vector
- 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)
- 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)
- 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)

## 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.

## Claims

### Claim 1

A polynucleotide encoding a multidomain therapeutic protein comprising a delivery domain and an enzyme domain,
wherein the delivery domain is an antigen-binding protein that binds to CD63, and wherein the antigen binding protein is a single-chain variable fragment (scFv),
wherein the enzyme domain comprises an alpha-glucosidase (GAA) or a portion thereof, and
wherein the polynucleotide further comprises a virus nucleic acid sequence, wherein the virus nucleic acid sequence is an adeno-associated virus (AAV) nucleic acid sequence.

### Claim 2 (depends on 1)

The polynucleotide of claim 1, wherein the polynucleotide comprises a locus-targeting nucleic acid sequence.

### Claim 3 (depends on 1)

The polynucleotide of claim 1, wherein the polynucleotide comprises a tissue-specific regulatory element.

### Claim 4 (depends on 3)

The polynucleotide of claim 3, wherein the tissue-specific regulatory element is a liver-specific promoter.

### Claim 5 (depends on 1)

The polynucleotide of claim 1, wherein the enzyme domain comprises the amino acid sequence set forth in SEQ ID NO: 1 or a biologically active portion thereof.

### Claim 6 (depends on 1)

The polynucleotide of claim 1, wherein the delivery domain comprises:
an amino acid sequence of SEQ ID NO: 2;
the HCDR-1-HCDR-2-HCDR3-LCDR1-LCDR2-LCDR-3 amino acid sequences contained within an HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOS: 14/22, SEQ ID NOS: 30/38, SEQ ID NOS: 46/54, and SEQ ID NOS: 62/70;
HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences selected from the group consisting of SEQ ID NOs: 16-18-20-24-26-28, SEQ ID NOs: 32-34-36-40-42-44, SEQ ID NOs: 48-50-52-56-58-60, and SEQ ID NOs: 64-66-68-72-74-76; or
an HCVR/LCVR amino acid pair set forth in SEQ ID NOS 14/22, SEQ ID NOS: 30/38, SEQ ID NOS: 46/54, or SEQ ID NOS: 62/70.

### Claim 7 (depends on 6)

The polynucleotide of claim 6, wherein the HCVR/LCVR amino acid pair comprises from N-terminal to C-terminal: the HCVR, a linker, and the LCVR.

### Claim 8 (depends on 7)

The polynucleotide of claim 7, wherein the enzyme domain is attached to the C-terminus of the LCVR via a linker.

### Claim 9 (depends on 8)

The polynucleotide of claim 8, wherein the delivery domain comprises the HCVR/LCVR amino acid pair set forth in SEQ ID NOS: 46/54.

### Claim 10 (depends on 1)

The polynucleotide of claim 1, wherein the multidomain therapeutic protein comprises the amino acid sequence set forth in SEQ ID NO: 79.

### Claim 11 (depends on 1)

A composition comprising a gene therapy vector comprising the polynucleotide of claim 1, wherein the gene therapy vector is an AAV vector comprising the polynucleotide.

### Claim 12 (depends on 11)

The composition of claim 11, wherein the gene therapy vector is an AAV2/8 chimera.

### Claim 13 (depends on 11)

The composition of claim 11, further comprising a pharmaceutically acceptable carrier.

### Claim 14 (depends on 1)

A recombinant multidomain therapeutic protein encoded by the polynucleotide of claim 1.

### Claim 15 (depends on 11)

A method of expressing in a cell a recombinant multidomain therapeutic protein, comprising:
(a) contacting the cell with the composition of claim 11;
(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.

### Claim 16 (depends on 15)

The method of claim 15, wherein the cell is a human cell.

### Claim 17 (depends on 15)

The method of claim 15, wherein the cell is in vivo.

### Claim 18 (depends on 15)

The method of claim 15, 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 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.

### Claim 19 (depends on 15)

The method of claim 15, wherein the enzyme domain comprises the amino acid sequence set forth in SEQ ID NO: 1 or a fragment thereof.

### Claim 20 (depends on 15)

The method of claim 15, wherein the delivery domain comprises:
an amino acid sequence of SEQ ID NO: 2;
the HCDR-1-HCDR-2-HCDR3-LCDR1-LCDR2-LCDR-3 amino acid sequences contained within an HCVR/LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOS: 14/22, SEQ ID NOS: 30/38, SEQ ID NOS: 46/54, and SEQ ID NOS: 62/70;
HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3 amino acid sequences selected from the group consisting of SEQ ID NOs: 16-18-20-24-26-28, SEQ ID NOs: 32-34-36-40-42-44, SEQ ID NOs: 48-50-52-56-58-60, and SEQ ID NOs: 64-66-68-72-74-76; or
an HCVR/LCVR amino acid pair set forth in SEQ ID NOS 14/22, SEQ ID NOS: 30/38, SEQ ID NOS: 46/54, or SEQ ID NOS: 62/70.

### Claim 21 (depends on 20)

The method of claim 20, wherein the HCVR/LCVR amino acid pair comprises from N-terminal to C-terminal: the HCVR, a linker, and the LCVR.

### Claim 22 (depends on 21)

The method of claim 21, wherein the enzyme domain is attached to the C-terminus of the LCVR via a linker.

### Claim 23 (depends on 22)

The method of claim 22, wherein the delivery domain comprises the HCVR/LCVR amino acid pair set forth in SEQ ID NOS: 46/54.

### Claim 24 (depends on 15)

The method of claim 15, wherein the multidomain therapeutic protein comprises the amino acid sequence set forth in SEQ ID NO: 79.