# US 2019/0167810 A1 · Exosomes comprising therapeutic polypeptides

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- Number: US 2019/0167810 A1
- Document ID: US20190167810A1
- Status: Published application
- Kind: A1
- Application number: 16/304,580
- Filed: 2017-05-25
- Published: 2019-06-06
- Earliest claimed: 2016-05-25
- Assignees: Evox Therapeutics Ltd; Oxford University Innovation Limited
- Applicants: Evox Therapeutics Ltd; Oxford University Innovation
- Inventors: Justin HEAN; Imre MAGER; Matthew WOOD; Samir EL ANDALOUSSI; Oscar WIKLANDER; Joel NORDIN
- Web page: https://patentgrep.com/patent/US20190167810A1

## CPC

- A61K 47/6901: {Conjugates being cells, cell fragments, viruses, ghosts, red blood cells or viral vectors}
- A61K 48/0008: {characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition}
- A61K 9/127: Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
- A61K 38/47: acting on glycosyl compounds (3.2), e.g. cellulases, lactases
- A61K 41/0042: {Photocleavage of drugs in vivo, e.g. cleavage of photolabile linkers in vivo by UV radiation for releasing the pharmacologically-active agent from the administered agent; photothrombosis or photoocclusion}
- A61K 47/00: Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K 47/6425: {the peptide or protein in the drug conjugate being a receptor, e.g. CD4, a cell surface antigen, i.e. not a peptide ligand targeting the antigen, or a cell surface determinant, i.e. a part of the surface of a cell}
- A61K 47/65: Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
- A61K 48/005: {characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered}
- A61P 1/16: for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
- A61P 3/06: Antihyperlipidemics
- A61P 3/10: for hyperglycaemia, e.g. antidiabetics
- A61P 7/06: Antianaemics
- A61P 9/00: Drugs for disorders of the cardiovascular system
- A61P 9/10: for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
- A61P 11/00: Drugs for disorders of the respiratory system
- A61P 13/12: of the kidneys
- A61P 15/00: Drugs for genital or sexual disorders (for disorders of sex hormones A61P5/24); Contraceptives
- A61P 17/00: Drugs for dermatological disorders
- A61P 17/06: Antipsoriatics
- A61P 19/02: for joint disorders, e.g. arthritis, arthrosis
- A61P 19/08: for bone diseases, e.g. rachitism, Paget's disease
- A61P 21/00: Drugs for disorders of the muscular or neuromuscular system
- A61P 21/04: for myasthenia gravis
- A61P 25/00: Drugs for disorders of the nervous system
- A61P 25/02: for peripheral neuropathies
- A61P 25/14: for treating abnormal movements, e.g. chorea, dyskinesia
- A61P 25/16: Anti-Parkinson drugs
- A61P 25/28: for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
- A61P 29/00: Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
- A61P 31/04: Antibacterial agents
- A61P 35/00: Antineoplastic agents
- A61P 37/02: Immunomodulators
- A61P 37/06: Immunosuppressants, e.g. drugs for graft rejection
- C12Y 302/01021: Beta-glucosidase (3.2.1.21)
- A61K 35/33: Fibroblasts
- A61K 48/0091: {Purification or manufacturing processes for gene therapy compositions}
- 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
- C12Y: ENZYMES

## Abstract

The present invention pertains to an inventive release mechanism for extracellular vesicle (EV)-mediated intracellular and intramembrane delivery of therapeutic polypeptides. More specifically, the invention relates to EVs comprising polypeptide constructs which comprise a therapeutic polypeptide releasably attached to an exosomal polypeptide. Furthermore, the present invention pertains to manufacturing methods, pharmaceutical compositions, medical uses and applications, and various other embodiments related to the inventive EVs.

## Claims

### Claim 1

An extracellular vesicle (EV) comprising at least one polypeptide construct comprising:
(i) at least one polypeptide of interest (PoI);
(ii) at least one exosomal polypeptide; and,
(iii) a polypeptide-based release system,
wherein the polypeptide-based release system releases the at least one PoI from the at least one exosomal polypeptide upon endogenous activation.

### Claim 2 (depends on 1)

The EV according to claim 1, wherein the polypeptide-based release system is selected from a cis-cleaving release system or a nuclear localization signal (NLS)—nuclear localization signal-binding protein (NLSBP) (NLS-NLSBP) release system.

### Claim 3 (depends on 1)

The EV according to claim 1, wherein the PoI is a therapeutic polypeptide selected from the group comprising antibodies, intrabodies, single chain variable fragments, affibodies, enzymes, tumor suppressors, viral or bacterial inhibitors, cell component proteins, DNA and/or RNA binding proteins, DNA repair inhibitors, nucleases, proteinases, integrases, transcription factors, growth factors, apoptosis inhibitors and inducers, toxins, structural proteins, neurotrophic factors, membrane transporters, nucleotide binding proteins, heat shock proteins, CRISPR-associated proteins, and any combination thereof.

### Claim 4 (depends on 1)

The EV according to claim 1, wherein the exosomal polypeptide is selected from the group comprising CD9, CD53, CD63, CD81, CD54, CD50, FLOT1, FLOT2, CD49d, CD71, CD133, CD138, CD235a, ALIX, Syntenin-1, 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, tetraspanins, Fc receptors, interleukin receptors, immunoglobulins, MHC-I or MHC-II components, 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, L1CAM, LAMB1, LAMC1, LFA-1, LGALS3BP, Mac-1 alpha, Mac-1 beta, MFGE8, SLIT2, STX3, TCRA, TCRB, TCRD, TCRG, VTI1A, VTI1B, and other exosomal polypeptides.

### Claim 5 (depends on 1)

The EV according to claim 1, wherein the EV further comprises at least one polynucleotide of interest.

### Claim 6 (depends on 5)

The EV according to claim 5, wherein the polynucleotide of interest is transported into the EV through interaction with an RNA-binding PoI or a DNA-binding PoI.

### Claim 7

An EV comprising at least one PoI, wherein the PoI has been released from at least one an exosomal polypeptide through endogenous activation of a polypeptide-based release system.

### Claim 8 (depends on 7)

The EV according to claim 7, wherein the EV further comprises a polynucleotide of interest.

### Claim 9 (depends on 1)

The EV according to claim 1, wherein the EV further comprises at least one targeting moiety.

### Claim 10 (depends on 1, 7)

A method for producing EVs according to claim 1 or 7, comprising:
i. introducing into an EV-producing cell at least one polynucleotide construct encoding at least one polypeptide construct comprising at least one PoI, an endogenously activatable polypeptide-based release system, and at least one exosomal polypeptide;
ii. expressing in the EV-producing cell at least one polypeptide construct encoded for by the at least one polynucleotide construct; and,
iii. collecting from the EV-producing cell EVs comprising a PoI.

### Claim 11

A polynucleotide construct encoding for:
i. at least one PoI;
ii. at least one endogenously activatable polypeptide-based release system; and,
iii. at least one exosomal polypeptide.

### Claim 12

A polypeptide construct comprising:
i. at least one PoI;
ii. at least one endogenously activatable polypeptide-based release system; and,
iii. at least one exosomal polypeptide.

### Claim 13 (depends on 12)

An EV comprising at least one polypeptide construct according to claim 12.

### Claim 14 (depends on 1, 7, 11, 12, 13)

A cell comprising (i) at least one polynucleotide construct according to claim 11 or (ii) at least one polypeptide construct according to claim 12 or (iii) at least one EV according to any one of claim 1, 7 or 13.

### Claim 15 (depends on 1, 7, 13)

An in vitro method for intracellular delivery of at least one PoI, comprising contacting a target cell with at least one EV according to any one of claim 1, 7 or 13.

### Claim 16 (depends on 1, 7, 11, 12, 13)

A pharmaceutical composition comprising at least one polynucleotide construct according to claim 11 or at least one polypeptide construct according to claim 12 or at least one EV according to any one of claim 1, 7 or 13.

### Claim 17

(canceled)

### Claim 18 (depends on 1, 7, 13)

A method of treating a disease or disorder comprising administering to a subject a therapeutically effective amount of an EV or a population of EVs according to any one of claim 1, 7 or 13.

### Claim 19 (depends on 3)

The EV of claim 3, wherein the PoI is an enzyme for enzyme replacement therapy or gene editing.

### Claim 20 (depends on 7)

The EV according to claim 7, wherein the EV further comprises at least one targeting moiety.

### Claim 21 (depends on 1, 15)

The in vitro method of claim 15, further comprising contacting the target cell with at least one EV according to claim 1.

### Claim 22 (depends on 7, 15)

The in vitro method of claim 15, further comprising contacting the target cell with at least one EV according to claim 7.