# US 11,708,569 B2 · Modified recombinant lysosomal alpha-galactosidase A and aspartylglucoaminidase having low mannose-6-phosphate and high sialic acid

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- Number: US 11,708,569 B2
- Document ID: US11708569B2
- Status: Granted
- Kind: B2
- Application number: 17/272,178
- Filed: 2019-08-29
- Published: 2022-04-28
- Granted: 2023-07-25
- 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/US11708569B2

## 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 less than 10% mannose-6-phosphate (Man6P) and less than 0.3 mole exposed mannose (Man) per mole of enzyme, and wherein said enzyme comprises more than 4 moles alpha2,3 sialic acid (SA) per mole of enzyme, and wherein the modified recombinant lysosomal enzyme is Alpha-Galactosidase A (GLA) or Aspartvlglucoaminidase (AGA).

### Claim 2 (depends on 1)

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

### Claim 3 (depends on 1)

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

### Claim 4 (depends on 1)

The modified recombinant lysosomal enzyme of claim 1, wherein the modified recombinant lysosomal enzyme comprises no detectable M6P, and high 2,3SA.

### Claim 5 (depends on 4)

The modified recombinant lysosomal enzyme of claim 4, 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 6 (depends on 1)

The modified recombinant lysosomal enzyme of claim 1, wherein said modified recombinant lysosomal enzyme comprises lowered mannose-6- phosphate (M6P) tagging of N-glycans as compared to a similar unmodified recombinant lysosomal enzyme.