# US 2025/0082684 A1 · Recombinant Cell Line Expressing Membrane Proteins and Vesicles Prepared Therefrom

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- Number: US 2025/0082684 A1
- Document ID: US20250082684A1
- Status: Published application
- Kind: A1
- Application number: 18/728,983
- Filed: 2023-01-17
- Published: 2025-03-13
- Earliest claimed: 2022-01-20
- Applicants: Exocure Sweden AB
- Inventors: Jan Lötvall; Kyong-su Park; Cecilia Lasser
- Web page: https://patentgrep.com/patent/US20250082684A1

## CPC

- A61K 35/28: Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
- C07K 14/705: Receptors; Cell surface antigens; Cell surface determinants {(tumour specific antigens C07K14/4748)}
- A61K 35/30: Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
- A61K 35/34: Muscles; Smooth muscle cells; Heart; Cardiac stem cells; Myoblasts; Myocytes; Cardiomyocytes (vascular smooth muscle A61K35/44)
- A61K 35/36: Skin; Hair; Nails; Sebaceous glands; Cerumen; Epidermis; Epithelial cells; Keratinocytes; Langerhans cells; Ectodermal cells (islets of Langerhans A61K35/39)
- A61K 35/545: Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
- A61P 29/00: Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
- A61P 37/06: Immunosuppressants, e.g. drugs for graft rejection
- C12N 5/0662: {Stem cells}
- A61K 2035/124: {the cells being hematopoietic, bone marrow derived or blood cells}
- C12N 2510/00: Genetically modified cells
- 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)
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present disclosure provides vesicles prepared from a mammalian cell line genetically modified to overexpress one or more membrane proteins as provided in the disclosure. The genetically modified cells are also provided. The vesicles find use in methods for reducing inflammatory response in a subject in need thereof.

## Claims

### Claim 1

A composition comprising:
vesicles prepared from a mammalian cell line genetically modified to overexpress a membrane protein selected from the group consisting of: bifunctional heparan sulfate N-deacetylase/N-sulfotransferase 1 (NDST1), ferroptosis suppressor protein 1 (AIFM2), cysteine-rich and transmembrane domain-containing protein 1 (CYSTM1), cadherin-6 (CDH6), solute carrier family 22 member 18 (SLC22A18), promethin (TMEM159), receptor-type tyrosine-protein phosphatase kappa (PTPRK), prolyl endopeptidase FAP (FAP), phospholipid-transporting ATPase ID (ATP8B2), ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase 2 (BST1), reticulon-2 (RTN2), ras-related protein Rap-1A (RAP1A), basigin (BSG), aldehyde dehydrogenase family 3 member B1 (ALDH3B1), calsyntenin-1 (CLSTN1), small VCP/p97-interacting protein (SVIP), GTP-binding protein Rheb (RHEB), lysophosphatidic acid receptor 1 (LPAR1), prolow-density lipoprotein receptor-related protein 1 (LRP1), receptor-type tyrosine-protein phosphatase F (PTPRF), raftlin (RFTN1), guanine nucleotide-binding protein G (q) subunit alpha (GNAQ), guanine nucleotide-binding protein subunit alpha-11 (GNA11), integrin alpha-8 (ITGA8), roundabout homolog 1 (ROBO1), ADP-ribosylation factor 6 (ARF6), ephrin type-B receptor 2 (EPHB2), transforming protein RhoA (RHOA), proto-oncogene tyrosine-protein kinase Src (SRC), synaptobrevin homolog YKT6 (YKT6), Ras-related protein Rab-23 (RAB23), vascular cell adhesion protein 1 (VCAM1), lymphocyte function-associated antigen 3 (CD58), guanine nucleotide-binding protein G (I)/G(S)/G (O) subunit gamma-12 (GNG12), 2′,3′-cyclic-nucleotide 3′-phosphodiesterase (CNP), receptor-type tyrosine-protein phosphatase eta (PTPRJ), guanine nucleotide-binding protein G(s) subunit alpha isoforms short (GNAS), ADP-ribosylation factor 5 (ARF5), mitochondrial calcium uniporter regulator 1 (MCUR1), nuclear mitotic apparatus protein 1 (NUMA1), protein/nucleic acid deglycase DJ-1 (PARK7), V-type immunoglobulin domain-containing suppressor of T-cell activation (VISTA), CD276, Cell surface glycoprotein MUC18 (MCAM), and Phosphatidylserine synthase 2 (PTDSS2); and
a pharmaceutically acceptable carrier,
wherein the vesicles when administered to a subject reduces the levels of at least one proinflammatory cytokine in the subject.

### Claim 2 (depends on 1)

The composition of claim 1, wherein the mammalian cell line is genetically modified to contain an exogenous nucleic acid sequence encoding the membrane protein.

### Claim 3 (depends on 2)

The composition of claim 2, wherein the exogenous nucleic acid sequence is operably linked to a heterologous promoter.

### Claim 4 (depends on 1, 2, 3)

The composition of any one of claims 1-3, wherein the mammalian cell line is genetically modified to overexpress at least two of the membrane proteins.

### Claim 5 (depends on 1, 2, 3)

The composition of any one of claims 1-3, wherein the mammalian cell line is genetically modified to overexpress three or more of the membrane proteins.

### Claim 6 (depends on 1, 2, 3, 4, 5)

The composition of any one of claims 1-5, wherein the composition comprises a first population of vesicles prepared from a first mammalian cell line genetically modified to overexpress a first membrane protein and a second population of vesicles prepared from a second mammalian cell line genetically modified to overexpress a second membrane protein, wherein the first and second membrane proteins are independently selected from the group consisting of: bifunctional heparan sulfate N-deacetylase/N-sulfotransferase 1, ferroptosis suppressor protein 1, cysteine-rich and transmembrane domain-containing protein, cadherin-6, solute carrier family 22 member 18, promethin, receptor-type tyrosine-protein phosphatase kappa, prolyl endopeptidase FAP, phospholipid-transporting ATPase ID, ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase 2, reticulon-2, ras-related protein Rap-1A, basigin, aldehyde dehydrogenase family 3 member B1, calsyntenin-1, small VCP/p97-interacting protein, GTP-binding protein Rheb, lysophosphatidic acid receptor 1, prolow-density lipoprotein receptor-related protein 1, receptor-type tyrosine-protein phosphatase F, raftlin, guanine nucleotide-binding protein G (q) subunit alpha, guanine nucleotide-binding protein subunit alpha-11, integrin alpha-8, roundabout homolog 1, ADP-ribosylation factor 6, ephrin type-B receptor 2, transforming protein RhoA, proto-oncogene tyrosine-protein kinase Src, synaptobrevin homolog YKT6, Ras-related protein Rab-23, vascular cell adhesion protein 1, lymphocyte function-associated antigen 3, guanine nucleotide-binding protein G (I)/G(S)/G (O) subunit gamma-12, 2′,3′-cyclic-nucleotide 3′-phosphodiesterase, receptor-type tyrosine-protein phosphatase eta, guanine nucleotide-binding protein G(s) subunit alpha isoforms short, ADP-ribosylation factor 5, mitochondrial calcium uniporter regulator 1, nuclear mitotic apparatus protein 1, protein/nucleic acid deglycase DJ-1, V-type immunoglobulin domain-containing suppressor of T-cell activation, CD276, MCAM and Phosphatidylserine synthase 2.

### Claim 7 (depends on 6)

The composition of claim 6, further comprising a third population of vesicles prepared from a third mammalian cell line genetically modified to overexpress a third membrane protein selected from the listed membrane proteins.

### Claim 8 (depends on 6)

The composition of claim 6, further comprising a plurality of populations of vesicles prepared from a plurality mammalian cell lines genetically modified to overexpress a membrane protein selected from the listed membrane proteins.

### Claim 9 (depends on 6)

The composition of claim 6, wherein the first and/or the second mammalian cell line is further genetically modified to overexpress a different membrane protein selected from the listed membrane proteins.

### Claim 10 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9)

The composition of any one of claims 1-9, wherein the mammalian cell line is generated from a parental cell, wherein the parental cell is a mammalian cell line or a primary cell.

### Claim 11 (depends on 10)

The composition of claim 10, wherein the parental cell is a mammalian cell line, wherein the mammalian cell line is HEK293 cell line, CHO cell line, or embryonic stem cell line.

### Claim 12 (depends on 10)

The composition of claim 10, wherein the parental cell is a primary cell, wherein the primary cell is a stem cell isolated from a subject.

### Claim 13 (depends on 12)

The composition of claim 12, wherein the stem cell is an embryonic stem cell, an induced pluripotent stem cell, a hematopoietic stem cell, a neuronal stem cell, a mesenchymal stem cell, a muscle stem cell, or a skin stem cell.

### Claim 14 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)

The composition of any one of claims 1-13, wherein the at least one proinflammatory cytokine comprises IL-2, IL-4, IL-6, IL-12, IL-12p70, IL-17, tumor necrosis factor alpha (TNF-α), or interferon gamma (IFN-γ).

### Claim 15 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)

The composition of any one of claims 1-14, wherein the vesicles comprise extracellular vesicles (EVs), nanovesicles (NVs), synthetic eukaryotic vesicles (SyEVs), or a mixture of two or more of EVs, NVs, and SyEVs.

### Claim 16

A mammalian cell, wherein the mammalian cell is genetically modified to overexpress a membrane protein selected from the group consisting of: bifunctional heparan sulfate N-deacetylase/N-sulfotransferase 1, ferroptosis suppressor protein 1, cysteine-rich and transmembrane domain-containing protein, cadherin-6, solute carrier family 22 member 18, promethin, receptor-type tyrosine-protein phosphatase kappa, prolyl endopeptidase FAP, phospholipid-transporting ATPase ID, ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase 2, reticulon-2, ras-related protein Rap-1A, basigin, aldehyde dehydrogenase family 3 member B1, calsyntenin-1, small VCP/p97-interacting protein, GTP-binding protein Rheb, lysophosphatidic acid receptor 1, prolow-density lipoprotein receptor-related protein 1, receptor-type tyrosine-protein phosphatase F, raftlin, guanine nucleotide-binding protein G (q) subunit alpha, guanine nucleotide-binding protein subunit alpha-11, integrin alpha-8, roundabout homolog 1, ADP-ribosylation factor 6, ephrin type-B receptor 2, transforming protein RhoA, proto-oncogene tyrosine-protein kinase Src, synaptobrevin homolog YKT6, Ras-related protein Rab-23, vascular cell adhesion protein 1, lymphocyte function-associated antigen 3, guanine nucleotide-binding protein G (I)/G(S)/G (O) subunit gamma-12, 2′,3′-cyclic-nucleotide 3′-phosphodiesterase, receptor-type tyrosine-protein phosphatase eta, guanine nucleotide-binding protein G(s) subunit alpha isoforms short, ADP-ribosylation factor 5, mitochondrial calcium uniporter regulator 1, nuclear mitotic apparatus protein 1, protein/nucleic acid deglycase DJ-1, V-type immunoglobulin domain-containing suppressor of T-cell activation, CD276, MCAM and Phosphatidylserine synthase 2.

### Claim 17 (depends on 16)

The mammalian cell line of claim 16, wherein the mammalian cell line is genetically modified to contain an exogenous nucleic acid sequence encoding the membrane protein.

### Claim 18 (depends on 17)

The mammalian cell line of claim 17, wherein the exogenous nucleic acid sequence is operably linked to a heterologous promoter.

### Claim 19 (depends on 16, 17, 18)

The mammalian cell line of any one of claims 16-18, wherein the mammalian cell line is genetically modified to overexpress at least two of the membrane proteins.

### Claim 20 (depends on 16, 17, 18)

The mammalian cell line of any one of claims 16-18, wherein the mammalian cell line is genetically modified to overexpress three or more of the membrane proteins.

### Claim 21 (depends on 16, 17, 18)

The mammalian cell line of any one of claims 16-18, wherein the mammalian cell line is generated from a parental cell, wherein the parental cell is a mammalian cell line or a primary cell.

### Claim 22 (depends on 21)

The mammalian cell line of claim 21, wherein the parental cell is a mammalian cell line, wherein the mammalian cell line is HEK293 cell line, CHO cell line, or embryonic stem cell line.

### Claim 23 (depends on 21)

The mammalian cell line of claim 21, wherein the parental cell is a primary cell, wherein the primary cell is a stem cell isolated from a subject.

### Claim 24 (depends on 23)

The mammalian cell line of claim 23, wherein the stem cell is an embryonic stem cell, an induced pluripotent stem cell, a hematopoietic stem cell, a neuronal stem cell, a mesenchymal stem cell, a muscle stem cell, or a skin stem cell.

### Claim 25 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)

A method for reducing inflammation in a subject in need thereof, the method comprising:
administering to the subject an effective amount of the composition of any one of claims 1-15,
wherein the administering reduces at least one proinflammatory cytokine in the subject.

### Claim 26 (depends on 25)

The method of claim 25, wherein the subject has or is susceptible to developing an inflammatory related condition selected from the group consisting of cancer, multiple sclerosis, psoriasis, dry eye disease, asthma, sepsis, infection, Rheumatoid arthritis, ulcerative colitis, Crohn's disease, tuberculosis, hepatitis, sinusitis, autoimmune disease, inflammatory bowel disease, pelvic inflammatory disease, ulcers, atherosclerosis, erythema, necrosis, vasculitis, ankylosing spondylitis, connective tissue disease, kidney disease, sarcoidosis, thyroiditis, osteoarthritis, Rheumatism, chronic inflammatory condition, demyelinating polyneuropathy, pancreatitis, psoriatic arthritis, periodontitis, Behcet's disease, sinusitis, polymyalgia rheumatic, nephritis, diverticulitis, granulomatosis with polyangilitis, granuloma, encephalitis, immune-mediated inflammatory disease, esophagitis, gout, uveitis, myopathy, gallbladder disease, periodic fever syndrome, interstitial cystitis, peritonitis, appendicitis, Parkinson's disease, Alzheimer's, systemic lupus erythematous, fibromyalgia, diverticulitis, dermatitis and ankylosing spondylitis.

### Claim 27 (depends on 26)

The method of claim 26, wherein the inflammatory related condition is inflammatory respiratory disease.

### Claim 28 (depends on 26)

The method of claim 26, wherein the inflammatory related condition is sepsis/cytokine storm.

### Claim 29 (depends on 26)

The method of claim 26, wherein the inflammatory related condition is infection.

### Claim 30 (depends on 29)

The method of claim 29, wherein the infection is a bacterial, viral or parasitic infection.

### Claim 32 (depends on 25, 26, 27, 28, 29, 30)

The method of any one of claims 25-30, wherein the method further comprises administering a therapeutic agent to the subject.

### Claim 33 (depends on 32)

The method of claim 32, wherein the method comprises administering the composition and the therapeutic agent to the subject.

### Claim 34 (depends on 32)

The method of claim 32, wherein the method comprises administering the composition to the subject, wherein the vesicles comprise the therapeutic agent.

### Claim 35 (depends on 32, 33, 34)

The method of any one of claims 32-34, wherein the therapeutic agent comprises a small molecule, a peptide, a nucleic acid, or a polypeptide.

### Claim 36 (depends on 32, 33, 34)

The method of any one of claims 32-34, wherein the administering comprises intravenous, subcutaneous, intramuscular, intraperitoneal, intraarterial, intraarticular, intracerebral (intraparenchymal) or intracerebroventricular administration.

### Claim 37 (depends on 32, 33, 34)

The method of any one of claims 32-34, wherein the administering comprises local administration to a target site in the subject.