# US 10,870,842 B2 · Stabilized alpha-galactosidase and uses thereof

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- Number: US 10,870,842 B2
- Document ID: US10870842B2
- Status: Granted
- Kind: B2
- Application number: 16/356,238
- Filed: 2019-03-18
- Published: 2019-08-22
- Granted: 2020-12-22
- Earliest claimed: 2009-11-17
- Assignees: Protalix Ltd.
- Applicants: Protalix Ltd.
- Inventors: Avidor Shulman; Ilya Ruderfer; Tehila Ben-Moshe; Talia Shekhter; Yaniv Azulay; Tali Kizhner; Yoseph Shaaltiel
- Web page: https://patentgrep.com/patent/US10870842B2

## CPC

- C12N 9/2465: {acting on alpha-galactose-glycoside bonds, e.g. alpha-galactosidase (3.2.1.22)}
- A61P 3/00: Drugs for disorders of the metabolism (of the blood or the extracellular fluid A61P7/00)
- C12Y 302/01022: Alpha-galactosidase (3.2.1.22)
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12Y: ENZYMES
- 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)

## Abstract

Multimeric protein structures comprising at least two alpha-galactosidase monomers being covalently linked to one another via a linking moiety are disclosed herein, as well a process for preparing same, and methods of treating Fabry disease via administration of a multimeric protein structure. The disclosed multimeric protein structures exhibit an improved performance, in terms of enhanced activity and/or a longer lasting activity under both lysosomal conditions and in a serum environment.

## Claims

### Claim 1

A stabilized form of α-galactosidase comprising at least two native α-galactosidase monomers covalently linked to one another via a linking moiety, wherein said α-galactosidase is a human α-galactosidase.

### Claim 2 (depends on 1)

The stabilized form of α-galactosidase of claim 1 comprising two native α-galactosidase monomers covalently linked to one another via a linking moiety.

### Claim 3 (depends on 1)

The stabilized form of α-galactosidase of claim 1, wherein the linking moiety is a non-peptidic moiety.

### Claim 4 (depends on 3)

The stabilized form of α-galactosidase of claim 3, wherein the linking moiety comprises poly(alkylene glycol) and at least two functional groups, wherein each functional group forms a covalent bond with one of the native α-galactosidase monomers.

### Claim 5 (depends on 4)

The stabilized form of α-galactosidase of claim 4, wherein the poly(alkylene glycol) comprises ethylene glycol and/or propylene glycol units linked together.

### Claim 6 (depends on 4)

The stabilized form of α-galactosidase of claim 4, wherein the linking moiety has the following general formula:
—X1—(CR1R2—CR3R4—Y)n-X2—
wherein:
each of X1 and X2 is a functional group that forms a covalent bond with at least one α-galactosidase monomer;
C is a carbon atom;
Y is an oxygen atom, a sulfur atom or NR5, wherein NR5 is a nitrogen atom attached to R5;
n is an integer from 5 to 150; and
each of R1, R2, R3, R4 and R5 is independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, alkoxy, hydroxy, oxo, thiol and thioalkoxy.

### Claim 7 (depends on 6)

The stabilized form of α-galactosidase of claim 6, wherein at least one of said functional groups forms an amide bond with an α-galactosidase monomer.

### Claim 8 (depends on 6)

The stabilized form of α-galactosidase of claim 6, wherein said linking moiety is at least 20 atoms long.

### Claim 9 (depends on 6)

The stabilized form of α-galactosidase of claim 6, wherein n is at least 25.

### Claim 10 (depends on 6)

The stabilized form of α-galactosidase of claim 6, wherein n is an integer from 40 to 70.

### Claim 11 (depends on 6)

The stabilized form of α-galactosidase of claim 6, wherein each of R1, R2, R3, R4 and R5 is independently selected from the group consisting of hydrogen and oxo.

### Claim 12 (depends on 1)

The stabilized form of α-galactosidase of claim 1, featuring a characteristic selected from the group consisting of:
(a) an α-galactosidase activity upon subjecting the stabilized form of α-galactosidase to human plasma conditions for one hour which is at least 10% higher than an activity of naturally occurring α-galactosidase upon subjecting said naturally occurring α-galactosidase to said human plasma conditions for one hour;
(b) an α-galactosidase activity which decreases upon subjecting the stabilized form of α-galactosidase to human plasma conditions for one hour by a percentage which is at least 10% less than the percentage by which an activity of naturally occurring α-galactosidase decreases upon subjecting said naturally occurring α-galactosidase to said human plasma conditions for one hour;
(c) an α-galactosidase activity which remains in a range of 50% to 150% of the initial activity upon subjecting the stabilized form of α-galactosidase to human plasma conditions for one hour;
(d) an α-galactosidase activity upon subjecting the stabilized form of α-galactosidase to lysosomal conditions for one week, which is at least 10% higher than an activity of naturally occurring α-galactosidase upon subjecting said naturally occurring α-galactosidase to said lysosomal conditions for one week;
(e) an α-galactosidase activity which decreases upon subjecting the stabilized form of α-galactosidase to lysosomal conditions for one day by a percentage which is at least 10% less than the percentage by which an activity of naturally occurring α-galactosidase decreases upon subjecting said naturally occurring α-galactosidase to said lysosomal conditions for one day;
(f) an α-galactosidase activity which remains in a range of 50% to 150% of the initial activity upon subjecting the stabilized form of α-galactosidase to lysosomal conditions for one day;
(g) an α-galactosidase activity immediately upon subjecting the stabilized form of α-galactosidase to lysosomal conditions, which is at least 10% higher than an activity of naturally occurring α-galactosidase immediately upon subjecting said naturally occurring α-galactosidase to said lysosomal conditions;
(h) an α-galactosidase activity immediately upon subjecting the stabilized form of α-galactosidase to an aqueous solution having a pH of 7 and a temperature of 37° C., which is at least 10% higher than an activity of naturally occurring α-galactosidase immediately upon subjecting said naturally occurring α-galactosidase to said aqueous solution having a pH of 7 and a temperature of 37° C.; and
(i) a circulating half-life in human plasma which is higher than a circulating half-life of naturally occurring α-galactosidase.

### Claim 13 (depends on 1)

The stabilized form of α-galactosidase of claim 1, wherein said human α-galactosidase is selected from the group consisting of agalsidase alpha and agalsidase beta.

### Claim 14 (depends on 1)

The stabilized form of α-galactosidase of claim 1, wherein said α-galactosidase is a plant recombinant α-galactosidase.

### Claim 15 (depends on 1)

A pharmaceutical composition comprising the stabilized form of α-galactosidase of claim 1 and a pharmaceutically acceptable carrier.

### Claim 16 (depends on 1)

A method of treating Fabry disease in a subject in need thereof, said method comprising administering to said subject the stabilized form of α-galactosidase of claim 1, such that said Fabry disease in said subject is treated.

### Claim 17 (depends on 16)

The method of claim 16, wherein said administering is by an intravenous infusion.

### Claim 18 (depends on 1)

A process of preparing the stabilized form of α-galactosidase of claim 1, said process comprising reacting α-galactosidase comprising at least two native α-galactosidase monomers with a cross-linking agent, wherein said cross-linking agent comprises a linking moiety and at least two reactive groups.

### Claim 19 (depends on 18)

The process of claim 18, wherein said reactive groups comprise a leaving group.

### Claim 20 (depends on 1)

The stabilized form of α-galactosidase of claim 1, wherein said human α-galactosidase comprises an amino acid sequence having at least 99% sequence homology to an amino acid sequence of an α-galactosidase naturally occurring in humans.