# US 12,325,871 B2 · Lysosomal enzymes modified by cell based glycoengineering

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- Number: US 12,325,871 B2
- Document ID: US12325871B2
- Status: Granted
- Kind: B2
- Application number: 18/199,575
- Filed: 2023-05-19
- Published: 2023-12-28
- Granted: 2025-06-10
- Earliest claimed: 2018-08-29
- Assignees: University of Copenhagen; Glycodisplay Aps
- Applicants: University of Copenhagen; Glycodisplay Aps
- Inventors: Claus Kristensen; Weihua Tian; Henrik Clausen; Zhang Yang; Sergey Vakhrushev
- Web page: https://patentgrep.com/patent/US12325871B2

## CPC

- C12N 9/2465: {acting on alpha-galactose-glycoside bonds, e.g. alpha-galactosidase (3.2.1.22)}
- C12N 9/2402: {hydrolysing O- and S- glycosyl compounds (3.2.1)}
- C12P 21/005: {Glycopeptides, glycoproteins}
- C12Y 302/01022: Alpha-galactosidase (3.2.1.22)
- C12Y 302/01045: Glucosylceramidase (3.2.1.45), i.e. beta-glucocerebrosidase
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C12Y: ENZYMES

## Abstract

The present invention relates to lysosomal enzymes modified by use of cell based methods, a compositions comprising a modified lysosomal enzyme, as well as methods for producing a modified lysosomal enzyme and therapeutic use of such modified lysosomal enzyme. In particular, the present disclosure relates to a modified lysosomal enzyme which has low Man6P and low exposed Mannose and high sialic acid content of alpha2,3 type enabling long circulation time and improved uptake into difficult-to-reach organs like heart, kidney and brain.

## Claims

### Claim 1

A modified recombinant lysosomal enzyme with increased circulation time in plasma as compared to an unmodified version of the same, wherein said enzyme comprises low mannose-6-phosphate (Man6P) and high sialic acid (SA) capping of alpha2,3 type (alpha2,3SA), wherein said enzyme comprises more than 4 mol alpha2,3SA per mol of enzyme.

### Claim 2 (depends on 1)

The modified recombinant lysosomal enzyme according to claim 1, wherein the enzyme comprises less than 0.3 mol Man6P per mol of enzyme.

### Claim 3 (depends on 1)

The modified recombinant lysosomal enzyme according to claim 1, wherein the enzyme comprises no detectable Man6P and high alpha2,3SA.

### Claim 4 (depends on 1)

The modified recombinant lysosomal enzyme according to claim 1, wherein the enzyme comprises less than 0.05 mol Man6P per mol of enzyme.

### Claim 5 (depends on 1)

The modified recombinant lysosomal enzyme according to claim 1, comprising more than 4.5 mol alpha2,3SA per mol of enzyme.

### Claim 6 (depends on 1)

The modified recombinant lysosomal enzyme according to claim 1, comprising more than 4 mol alpha2,3SA per mol of enzyme and less than 1 mol alpha2,6SA.

### Claim 7 (depends on 1)

The modified recombinant lysosomal enzyme according to claim 1, wherein said modified recombinant lysosomal enzyme comprises lowered Man6P tagging of N-glycans as compared to a similar unmodified recombinant lysosomal enzyme.

### Claim 8 (depends on 1)

The modified recombinant lysosomal enzyme according to claim 1, wherein the modified recombinant lysosomal enzyme comprises a biantennary N glycan structure, a triantennary N glycan structure, a tetra antennary N glycan structure, or combinations thereof.

### Claim 9 (depends on 1)

The modified recombinant lysosomal enzyme of claim 1, wherein the enzymes comprises low exposed Man6P.

### Claim 10 (depends on 1)

The modified recombinant lysosomal enzyme of claim 1, wherein the enzymes comprises less than 0.3 mol of exposed Man6P per mol of enzyme.

### Claim 11 (depends on 1)

The modified recombinant lysosomal enzyme according to claim 1, wherein the modified recombinant lysosomal enzyme is selected from Aspartylglucoaminidase (AGA), Alpha-Galactosidase A (GLA), Acid ceramidase, Acid alpha-L-fucosidase, Protective protein/Cathepsin A, Acid beta-glucosidase, or glucocerebrosidase (GBA), Acid beta-galactosidase, Iduronate-2-sulfatase (IDS), Alpha-LIduronidase (IDUA), Galactocerebrosidase/galactosylceramidase (GALC), Acid alphamannosidase, Acid beta-mannosidase, Arylsulfatase B, Arylsulfatase A, Acid betagalactosidase, N-Acetylglucosamine-1-phosphotransferase, and Lysosomal alpha-glucosidase (GAA).

### Claim 12 (depends on 1)

The modified recombinant lysosomal enzyme according to claim 1, wherein the modified recombinant lysosomal enzyme is selected from GLA, GBA, GUS, and GAA.

### Claim 13 (depends on 1)

A composition comprising a substantially pure preparation of the modified recombinant lysosomal enzyme according to claim 1.