# US 2020/0399591 A1 · Protein engineered extracellular vesicles

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- Number: US 2020/0399591 A1
- Document ID: US20200399591A1
- Status: Published application
- Kind: A1
- Application number: 16/763,145
- Filed: 2018-11-13
- Published: 2020-12-24
- Earliest claimed: 2017-11-13
- Applicants: Evox Therapeutics Ltd
- Inventors: Justin HEAN; Per LUNDIN; Andre GORGENS
- Web page: https://patentgrep.com/patent/US20200399591A1

## CPC

- C12N 5/0605: {Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly}
- A61K 9/5068: {Cell membranes or bacterial membranes enclosing drugs (liposomes with additional exogenous lipids A61K9/127)}
- A61K 31/445: Non condensed piperidines, e.g. piperocaine
- A61K 31/4545: containing a six-membered ring with nitrogen as a ring hetero atom, e.g. pipamperone, anabasine
- A61K 31/70: Carbohydrates; Sugars; Derivatives thereof (sorbitol A61K31/047)
- A61K 31/7016: Disaccharides, e.g. lactose, lactulose (lactobionic acid A61K31/7032)
- A61K 31/724: Cyclodextrins
- A61K 31/727: Heparin; Heparan
- A61K 35/28: Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
- A61K 35/50: Placenta; Placental stem cells; Amniotic fluid; Amnion; Amniotic stem cells
- A61P 3/00: Drugs for disorders of the metabolism (of the blood or the extracellular fluid A61P7/00)
- C07K 14/47: from mammals
- C07K 14/70596: {Molecules with a "CD"-designation not provided for elsewhere}
- C07K 14/79: Transferrins, e.g. lactoferrins, ovotransferrins
- C07K 2319/00: Fusion polypeptide
- C07K 2319/01: containing a localisation/targetting motif
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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
- 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)

## Abstract

The present invention relates to extracellular vesicles (EVs) as a novel therapeutic approach to lysosomal storage disorders (LSD). More specifically, the invention relates to the use of various protein engineering strategies for improving loading of hard-to-load LSD-related proteins and targeting of the resultant EVs to tissues and organs of interest.

## Claims

### Claim 1

Extracellular vesicles (EVs) obtainable from mesenchymal stromal cells (MSCs), amnion epithelial (AE) cells or placenta-derived cells, wherein the EVs comprise a plurality of polypeptide constructs comprising at least one lysosomal protein.

### Claim 2 (depends on 1)

The EVs according to claim 1, wherein the polypeptide construct further comprises at least one EV enrichment polypeptide.

### Claim 3 (depends on 1)

The EVs according to claim 1, wherein the lysosomal protein is selected from the group comprising alpha-D-mannosidase, N-aspartyl-beta-glucosaminidase, lysosomal acid lipase, cystinosin, lysosomal associated membrane protein-2, alpha-galactosidase A, acid ceramidase, alpha-fucosidase, cathepsin A, acid beta-glucosidase, beta-galactosidase, beta-hexosaminidase A, beta-hexosaminidase B, GlcNAc-1-phosphotransferase, beta-galactosylceramidase, lysosomal acid lipase, arylsulfatase A, alpha-L-iduronidase, iduronate-2-sulphatase, paran sulphamidase, acetyl alpha-glucosaminidase, acetyl CoA: alpha-glucosaminide-N-acetyltransferase, N-acetyl glucosamine-6-sulfatase, N-acetyl galactosamine-6-sulfatase, hyaluronidase, acetyl galactosamine-4-sulphatase, beta-glucuronidase, alpha-N-acetyl neuraminidase, N-actiylglucosamine-1-phosphotransferase, mucolipin-1, formylglycine-generating enzyme, palmitoyl-protein thioesterase-1, tripeptidyl peptidase I, cysteine string protein, CLN3p, CLN5p, CLN6p, CLN7p, CLN8p, acid sphingomyelinase, NPC 1, NPC 2, acid alpha-glucosidase, cathepsin K, sialin, alpha-N-acetylgalactosaminidase, GM2 activator, lysosomal acid lipase, and derivatives, regions, domains and/any combinations thereof.

### Claim 4 (depends on 1)

The EVs according to claim 1, wherein the EV enrichment polypeptide is selected from the group comprising CD9, CD53, CD63, CD81, CD54, CD50, FLOT1, FLOT2, CD49d, CD71, CD133, CD138, CD235a, ALIX, Syntenin (SEQ ID NO 1), the N terminal portion of syntenin (SEQ ID NO 2), Syntenin-2, Lamp2b, TSPAN8, TSPAN14, CD37, CD82, CD151, CD231, CD102, NOTCH1, NOTCH2, NOTCH3, NOTCH4, DLL1, DLL4, JAG1, JAG2, CD49d/ITGA4, ITGB5, ITGB6, ITGB7, CD11a, CD11b, CD11c, CD18/ITGB2, CD41, CD49b, CD49c, CD49e, CD51, CD61, CD104, Fc receptors, interleukin receptors, immunoglobulins, CD2, CD3 epsilon, CD3 zeta, CD13, CD18, CD19, CD30, CD34, CD36, CD40, CD40L, CD44, CD45, CD45RA, CD47, CD86, CD110, CD111, CD115, CD117, CD125, CD135, CD184, CD200, CD279, CD273, CD274, CD362, COL6A1, AGRN, EGFR, GAPDH, GLUR2, GLUR3, HLA-DM, HSPG2, Hsp70, L1CAM, LAMB1, LAMC1, LFA-1, LGALS3BP, Mac-1 alpha, Mac-1 beta, MFGE8, SLIT2, STX3, TCRA, TCRB, TCRD, TCRG, VTI1A, VTI1B, any derivatives and/or domains thereof, and any combinations thereof.

### Claim 5 (depends on 2)

The EVs according to claim 2, wherein the EV enrichment polypeptide is syntenin, the N-terminal part of syntenin, or CD63.

### Claim 6 (depends on 1)

The EVs according to claim 1, wherein the lysosomal protein is a lysosomal membrane protein.

### Claim 7 (depends on 1)

The EVs according to claim 1, wherein the EVs are positive for one or more of the following polypeptides: CD63, CD81, CD44, SSEA4, CD133, CD24, and heat shock 70 kDa protein 8.

### Claim 8 (depends on 1)

The EVs according to claim 1, wherein the EVs further comprise at least one heat shock protein.

### Claim 9 (depends on 8)

The EVs according to claim 8, wherein the at least one heat shock protein is heat shock 70 kDa protein 8.

### Claim 10 (depends on 1)

The EVs according to claim 1, further comprising a tissue targeting peptide and/or polypeptide.

### Claim 11 (depends on 10)

The EVs according to claim 10, wherein the tissue targeting peptide and/or polypeptide are comprised in the polypeptide construct and/or is present as a separate polypeptide construct.

### Claim 12 (depends on 1)

The EVs according to claim 1, wherein the polypeptide constructs comprising at least one lysosomal protein are substantially correctly folded.

### Claim 13 (depends on 1)

The EVs according to claim 1, wherein a single EV comprises:
(i) at least 10 copies of the polypeptide construct;
(ii) at least 50 copies of the polypeptide construct; and/or
(iii) at least 100 copies of the polypeptide construct.

### Claim 14 (depends on 1)

A method for producing EVs according to claim 1, comprising the steps of (i) introducing into an EV-producing MSC, AE or placenta-derived cell a polynucleotide construct encoding for a polypeptide construct comprising at least one lysosomal protein, and (ii) obtaining from the EV-producing cell EVs comprising a plurality of polypeptide constructs comprising at least one lysosomal protein.

### Claim 15 (depends on 14)

The method according to claim 14, wherein the EVs comprising a plurality of polypeptide constructs comprising at least one lysosomal protein comprise:
(i) at least 10 copies of the lysosomal protein;
(ii) at least 50 copies of the lysosomal protein; and/or
(iii) at least 100 copies of the lysosomal protein.

### Claim 16 (depends on 14)

The method according to claim 14, wherein the polypeptide constructs comprising at least one lysosomal protein also comprise at least one EV enrichment polypeptide.

### Claim 17

An EV-producing MSC, AE or placenta-derived cell comprising at least one polypeptide construct comprising at least one lysosomal protein, at least one polynucleotide construct encoding at least one lysosomal protein, and/or at least one vector comprising a polynucleotide construct encoding at least one lysosomal protein.

### Claim 18 (depends on 17)

The EV-producing MSC, AE or placenta-derived cell according to claim 17, wherein a polynucleotide construct encoding for a polypeptide construct comprising at least one lysosomal protein, and/or a vector comprising such a construct, is stably inserted into the EV-producing cell.

### Claim 19 (depends on 17)

The EV-producing MSC, AE or placenta-derived cell according to claim 17, wherein the polypeptide constructs comprising at least one lysosomal protein also comprise at least one EV enrichment polypeptide.

### Claim 20 (depends on 17)

The EV-producing MSC, AE or placenta-derived cell according to claim 17, wherein:
(i) at least 50%;
(ii) at least 70%; and/or
(iii) at least 90%;
of the EVs produced by the cell comprises the polypeptide construct comprising at least one lysosomal protein.

### Claim 21 (depends on 17)

The EV-producing MSC, AE or placenta-derived cell according to claim 17, wherein the EVs produced by the cell comprise:
(i) at least 10 copies of the lysosomal protein;
(ii) at least 50 copies of the lysosomal protein; and/or
(iii) at least 100 copies of the polypeptide construct comprising the lysosomal protein.

### Claim 22 (depends on 17)

The EV-producing MSC, AE or placenta-derived cell according to claim 17, wherein the cell is immortalized.

### Claim 23 (depends on 17)

The EV-producing MSC, AE or placenta-derived cell according to claim 17, wherein the cells and the EVs produced by the cells comprise one or more of the following native proteins: CD63, CD81, CD44, CD49e, CD105, SSEA4, CD133, CD24, and/or heat shock 70 kDa protein 8.

### Claim 24 (depends on 17)

The EV-producing MSC, AE or placenta-derived cell according to claim 17, wherein the cells and the EVs produced by the cells comprise the following native proteins: CD63, CD81, and heat shock 70 kDa protein 8.

### Claim 25 (depends on 1)

A pharmaceutical composition comprising a plurality of EVs according to claim 1, and a pharmaceutically acceptable carrier.

### Claim 26 (depends on 25)

The pharmaceutical composition according to claim 25, further comprising miglustat, arimoclomol, heparin, trehalose, and/or cyclodextrin and/or derivatives thereof.

### Claim 27 (depends on 1)

The EVs according to claim 1, for use in treating one or more lysosomal storage disorder.