# US 2019/0314409 A1 · Cancer-killing cells

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- Number: US 2019/0314409 A1
- Document ID: US20190314409A1
- Status: Published application
- Kind: A1
- Application number: 16/345,366
- Filed: 2017-10-25
- Published: 2019-10-17
- Earliest claimed: 2016-10-26
- Assignees: LIfT BioSciences Ltd
- Applicants: Lift Biosciences Ltd
- Inventors: Alex Blyth; Nico Bruyniks
- Web page: https://patentgrep.com/patent/US20190314409A1

## CPC

- A61K 35/15: Cells of the myeloid line, e.g. granulocytes, basophils, eosinophils, neutrophils, leucocytes, monocytes, macrophages or mast cells; Myeloid precursor cells; Antigen-presenting cells, e.g. dendritic cells (presenting a specific antigen A61K39/00; therapeutic combinations of antibodies, or fragments thereof, and blood-derived cells A61K39/00)
- A61K 31/198: Alpha-amino acids, e.g. alanine or edetic acid [EDTA] (betaine A61K31/205; proline A61K31/401; tryptophan A61K31/405; histidine A61K31/4172; peptides not degraded to individual amino acids A61K38/00)
- A61K 31/202: having three or more double bonds, e.g. linolenic (eicosanoids, e.g. leukotrienes A61K31/557)
- A61K 31/375: Ascorbic acid, i.e. vitamin C; Salts thereof
- A61K 31/59: Compounds containing 9, 10- seco- cyclopenta[a]hydrophenanthrene ring systems
- A61K 35/28: Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
- A61K 38/193: {Colony stimulating factors [CSF]}
- A61K 38/20: Interleukins [IL]
- A61K 38/27: Growth hormone [GH], i.e. somatotropin
- A61K 40/10: characterised by the cell type used
- A61K 40/42: Cancer antigens
- A61P 35/00: Antineoplastic agents
- C12N 5/0642: {Granulocytes, e.g. basopils, eosinophils, neutrophils, mast cells}
- C12N 5/0647: {Haematopoietic stem cells; Uncommitted or multipotent progenitors}
- G01N 33/57525: of the liver or pancreas
- A61K 2239/31: characterized by the route of administration
- A61K 2239/38: characterised by the dose, timing or administration schedule
- A61K 2239/54: Pancreas
- C12N 2500/32: Amino acids
- C12N 2500/38: Vitamins
- C12N 2501/02: Compounds of the arachidonic acid pathway, e.g. prostaglandins, leukotrienes
- C12N 2501/145: Thrombopoietin [TPO]
- C12N 2501/22: Colony stimulating factors (G-CSF, GM-CSF)
- C12N 2501/2303: Interleukin-3 (IL-3)
- C12N 2501/2304: Interleukin-4 (IL-4)
- C12N 2501/2306: Interleukin-6 (IL-6)
- C12N 2501/2308: Interleukin-8 (IL-8)
- C12N 2501/2318: Interleukin-18 (IL-18)
- C12N 2501/25: Tumour necrosing factors [TNF]
- C12N 2501/26: Flt-3 ligand (CD135L, flk-2 ligand)
- C12N 2501/305: Growth hormone [GH], aka. somatotropin
- C12N 2501/825: Serotonine (5-HT); Melatonine
- C12N 2501/998: Proteins not provided for elsewhere
- C12N 2506/11: from blood or immune system cells
- C12N 2506/45: from artificially induced pluripotent stem 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)
- 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)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

The present invention relates to an in vitro culture of haematopoietic cells, wherein said haematopoietic cells differentiate to form granulocytes characterised by the ability to kill cancer cells. The invention also relates to said granulocytes, methods for identifying said haematopoietic cells and granulocytes, compositions and kits comprising the same, as well as uses of the same for treating cancer.

## Claims

### Claim 1

An in vitro cell culture of haematopoietic cells, wherein said haematopoietic cells differentiate to form granulocytes characterised by:
a. a surface potential defined by an electrophoretic mobility of at least 1.0 μm·cm/volt·sec; and
b. the ability to kill cancer cells.

### Claim 2

An in vitro cell culture of haematopoietic cells, wherein said haematopoietic cells differentiate to form granulocytes characterised by:
a. a density of at least 1.077 g/ml; and
b. the ability to kill cancer cells.

### Claim 3

An in vitro cell culture of haematopoietic cells, wherein said haematopoietic cells differentiate to form granulocytes characterised by:
a. expression or activity of toll-like receptors; and/or an absence of expression or inactivity of: programmed death 1 (PD-1) receptor; CD115; CD224; CXCR1; and/or CXCR2; and
b. the ability to kill cancer cells.

### Claim 4 (depends on 1)

An in vitro cell culture according to claim 1, wherein the haematopoietic cells differentiate to form granulocytes further characterised by:
a. a density of at least 1.077 g/ml; and/or
b. expression or activity of toll-like receptors; and/or an absence of expression or inactivity of: programmed death 1 (PD-1) receptor; CD115; CD224; CXCR1; and/or CXCR2.

### Claim 5 (depends on 3, 4)

An in vitro cell culture according to claim 3 or 4, wherein the granulocytes are characterised by expression or activity of toll-like receptors; and an absence of expression or inactivity of: programmed death 1 (PD-1) receptor; CD115; CD224; CXCR1; and CXCR2.

### Claim 6

An in vitro cell culture according to any one of the preceding claims, wherein the cell culture is enriched for the haematopoietic cells.

### Claim 7

An in vitro cell culture according to any one of the preceding claims, wherein at least 70% of the cells in the in vitro cell culture are the haematopoietic cells.

### Claim 8

An in vitro cell culture according to any one of the preceding claims, wherein the haematopoietic cells are obtainable from a donor, preferably a human donor.

### Claim 9 (depends on 8)

An in vitro cell culture according to claim 8, wherein the donor is a male donor.

### Claim 10 (depends on 8, 9)

An in vitro cell culture according to claim 8 or 9, wherein the donor is aged 18 to 25.

### Claim 11 (depends on 1, 2)

An in vitro cell culture according to any one of the preceding claims, wherein the haematopoietic cells have a greater surface potential than otherwise identical haematopoietic cells that differentiate to form granulocytes having a surface potential defined by an electrophoretic mobility of less than 1.0 μm·cm/volt·sec and/or a reduced ability to kill cancer cells, when compared to feature b defined in claim 1, 2, or 3.

### Claim 12

An in vitro cell culture according to any one of the preceding claims, wherein the haematopoietic cells have a surface potential defined by an electrophoretic mobility of at least 1.0 μm·cm/volt·sec or at least 2.0 μm·cm/volt·sec or at least 2.5 μm·cm/volt·sec or at least 3.0 μm·cm/volt·sec.

### Claim 13

An in vitro cell culture according to any one of the preceding claims, wherein the granulocytes have a surface potential defined by an electrophoretic mobility of at least 2.0 μm·cm/volt·sec or at least 2.5 μm·cm/volt·sec or at least 3.0 μm·cm/volt·sec.

### Claim 14

An in vitro cell culture according to any one of the preceding claims, wherein the granulocyte is a neutrophil.

### Claim 15

A method for selecting a haematopoietic cell suitable for use in treating cancer, said method comprising:
a. measuring a surface potential of a granulocyte cell obtainable from a donor; and
b. selecting a haematopoietic cell from said donor when the measured surface potential is defined by an electrophoretic mobility of at least 1.0 μm·cm/volt·sec.

### Claim 16

A method for selecting a haematopoietic cell suitable for use in treating cancer, said method comprising:
a. measuring the density of a granulocyte cell obtainable from a donor; and
b. selecting a haematopoietic cell from said donor when the measured density of the granulocyte is at least 1.077 g/ml.

### Claim 17

A method for selecting a haematopoietic cell suitable for use in treating cancer, said method comprising:
a. detecting the expression or activity of toll-like receptors; programmed death 1 (PD-1) receptor; CD115; CD224; CXCR1; and/or CXCR2 on a granulocyte cell obtainable from a donor; and
b. selecting a haematopoietic cell from said donor when the toll-like receptors are expressed or active; and/or programmed death 1 (PD-1) receptor; CD115; CD224; CXCR1; and/or CXCR2 are not expressed or are inactive.

### Claim 18

A method for selecting a haematopoietic cell suitable for use in treating cancer, said method comprising:
a. measuring a surface potential of the haematopoietic cell; and
b. selecting a haematopoietic cell that has a greater surface potential than an otherwise identical haematopoietic cell that differentiates to form a granulocyte having a surface potential defined by an electrophoretic mobility of less than 1.0 μm·cm/volt·sec and/or has a reduced ability to kill cancer cells.

### Claim 19

A method for selecting a haematopoietic cell suitable for use in treating cancer, said method comprising:
a. measuring the density of a haematopoietic cell; and
b. selecting a haematopoietic cell that has a density greater than an otherwise identical haematopoietic cell that differentiates to form a granulocyte having a density of less than 1.077 g/ml and/or has a reduced ability to kill cancer cells

### Claim 20 (depends on 15, 16, 17, 18, 19)

A method according to any one of claims 15-19, wherein the haematopoietic cell has a surface potential defined by an electrophoretic mobility of at least 1.0 μm·cm/volt·sec.

### Claim 21 (depends on 15, 16, 17, 18, 19, 20)

A method according to any one of claims 15-20, wherein the haematopoietic cell has a surface potential defined by an electrophoretic mobility of at least 2.0 μm·cm/volt·sec or at least 2.5 μm·cm/volt·sec or at least 3.0 μm·cm/volt·sec.

### Claim 22 (depends on 15, 16, 17, 18, 19, 20, 21)

A method according any one of claims 15-21, wherein the surface potential is determined by electrophoresis.

### Claim 23 (depends on 15, 16, 17, 18, 19, 20, 21, 22)

A method according to any one of claims 15-22 further comprising discarding haematopoietic cells that are not selected in step b. of any one of claims 15-19.

### Claim 24 (depends on 15, 16, 17, 18, 19, 20, 21, 22, 23)

A method according to any one of claims 15-23, wherein the haematopoietic cell is a haematopoietic stem cell.

### Claim 25 (depends on 15, 16, 17, 18, 19, 20, 21, 22, 23, 24)

A method according to any one of claims 15-24, wherein the haematopoietic cell is a granulocyte precursor cell, such as a common myeloid progenitor cell, a myeloblast, a N. promyelocyte, a N. myelocyte, a N. metamyelocyte, a N. band, or combinations thereof.

### Claim 26 (depends on 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25)

A method according to any one of the claims 15-25, wherein the granulocyte is a neutrophil.

### Claim 27 (depends on 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26)

A method according to any one of claims 15-26 further comprising differentiating the haematopoietic cell into a granulocyte.

### Claim 28 (depends on 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27)

A method according to any one of claims 16-27, wherein the haematopoietic cell is obtainable from a donor, preferably a human donor.

### Claim 29 (depends on 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28)

A method according to any one of claims 15-28, wherein the donor is a male donor.

### Claim 30 (depends on 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29)

A method according to any one of claims 15-29, wherein the donor is aged 18 to 25.

### Claim 31

An in vitro method for selecting a haematopoietic cell suitable for use in treating cancer, said method comprising:
a. admixing a granulocyte obtainable from a donor with a cell line to form an admixture;
b. incubating said admixture;
c. measuring the % of cancer cells killed in said admixture; and
d. selecting a haematopoietic cell from said donor when said granulocyte kills at least 70% of the cancer cells in the admixture.

### Claim 32

Use of a surface potential of a haematopoietic cell, for selecting a cell that can be differentiated into a granulocyte that is suitable for treating cancer, wherein the surface potential is greater than the surface potential of an otherwise identical haematopoietic cell that differentiates to form a granulocyte having a surface potential defined by an electrophoretic mobility of less than 1.0 μm·cm/volt·sec and/or has a reduced ability to kill cancer cells.

### Claim 33 (depends on 32)

Use according to claim 32, wherein the haematopoietic cell has a surface potential defined by an electrophoretic mobility of at least 1.0 μm·cm/volt·sec, or at least 2.0 μm·cm/volt·sec, or at least 2.5 μm·cm/volt·sec, or at least 3.0 μm·cm/volt·sec.

### Claim 34

An in vitro method for selecting a granulocyte suitable for use in treating pancreatic cancer, said method comprising:
a. admixing a granulocyte with a pancreatic cancer cell line to form an admixture;
b. incubating said admixture;
c. measuring the % of pancreatic cancer cells killed in said admixture; and
d. selecting a granulocyte that kills at least 70% of the pancreatic cancer cells in the admixture.

### Claim 35 (depends on 34)

An in vitro method according to claim 34, wherein the pancreatic cancer cell line is a pancreatic ductal adenocarcinoma cell line.

### Claim 36

An in vitro method for selecting a granulocyte suitable for use in treating cancer, said method comprising:
a. admixing a granulocyte with a plurality of different cancer cell lines to provide a plurality of admixtures;
b. incubating said admixtures;
c. measuring the % of cancer cells killed in said admixtures; and
d. selecting a granulocyte as suitable for use in treating a cancer of the same type/subset as the cancer cell line, when said granulocyte kills at least 70% of the cancer cells in the admixture.

### Claim 37 (depends on 34, 35, 36)

An in vitro method according to any one of claims 34-36 further comprising discarding granulocytes that kill less than 70% of the cancer cells in the admixture.

### Claim 38 (depends on 34, 35, 36, 37)

An in vitro method according to any one of claims 34-37, wherein the granulocyte is obtainable from a donor, preferably a human donor.

### Claim 39 (depends on 34, 35, 36, 37, 38)

An in vitro method according to any one of claims 34-38, wherein the granulocyte is obtainable from a subject having a cancer of a different type/subset to the cancer cell line(s) used in the method.

### Claim 40 (depends on 34, 35, 36, 37, 38, 39)

An in vitro method according to claim any one of claims 34-39, wherein the cancer cell line(s) is one or more selected from: a pancreatic cancer cell line, a liver cancer cell line, an oesophageal cancer cell line, a stomach cancer cell line, a cervical cancer cell line, an ovarian cancer cell line, a lung cancer cell line, a bladder cancer cell line, a kidney cancer cell line, a brain cancer cell line, a prostate cancer cell line, a myeloma cancer cell line, a non-Hodgkin's lymphoma (NHL) cell line, a larynx cancer cell line, a uterine cancer cell line, or a breast cancer cell line.

### Claim 41 (depends on 38, 39, 40)

An in vitro method according to any one of claims 38-40, further comprising obtaining a haematopoietic cell from the donor from whom the selected granulocyte is obtainable.

### Claim 42 (depends on 34, 35, 36, 37, 38, 39, 40, 41)

A granulocyte obtainable by a method according to any one of claims 34-41.

### Claim 43 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31)

A method comprising differentiating an in vitro cell culture of haematopoietic cells according to any one of claims 1-14, or haematopoietic cells obtainable according to a method of any one of claims 15-31 into granulocytes.

### Claim 44 (depends on 43)

An in vitro cell culture of granulocytes obtainable by a method of claim 43, wherein said cell culture is enriched with granulocytes having:
a. a surface potential defined by an electrophoretic mobility of at least 1.0 μm·cm/volt·sec; and
b. the ability to kill cancer cells.

### Claim 45 (depends on 43)

An in vitro cell culture of granulocytes obtainable by a method of claim 43, wherein said cell culture is enriched with granulocytes having:
a. a density of at least 1.077 g/ml; and
b. the ability to kill cancer cells.

### Claim 46 (depends on 43)

An in vitro cell culture of granulocytes obtainable by a method of claim 43, wherein said cell culture is enriched with granulocytes having:
a. expression or activity of toll-like receptors; and/or an absence of expression or inactivity of: programmed death 1 (PD-1) receptor; CD115; CD224; CXCR1; and/or CXCR2; and
b. the ability to kill cancer cells.

### Claim 47

A pharmaceutical composition comprising:
a. a haematopoietic cell or a granulocyte; and
b. a granulocyte-macrophage colony-stimulating factor (GM-CSF), a granulocyte colony-stimulating factor (G-CSF), a growth hormone; serotonin, vitamin C, vitamin D, glutamine (Gln), arachidonic acid, AGE-albumin, an interleukin, TNF-alpha, Flt-3 ligand, thrombopoietin, foetal bovine serum (FBS), or combinations thereof.

### Claim 48 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 42, 44, 45, 46, 47)

An in vitro cell culture of haematopoietic cells according to any one of claims 1-14, or a granulocyte according to claim 42, or an in vitro cell culture of granulocytes according to claim 44-46, or a pharmaceutical composition according to claim 47, for use in treating cancer.

### Claim 49 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 42, 44, 45, 46, 47)

Use of an in vitro cell culture of haematopoietic cells according to any one of claims 1-14, or a granulocyte according to claim 42, or an in vitro cell culture of granulocytes according to any one of claims 44-46, or a pharmaceutical composition according to claim 47, in the manufacture of a medicament for treating cancer.

### Claim 50 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 42, 44, 45, 46, 47)

A method for treating cancer comprising: administering to a subject in need thereof an in vitro cell culture of haematopoietic cells according to any one of claims 1-14, or a granulocyte according to claim 42, or an in vitro cell culture of granulocytes according to any one of claims 44-46, or a pharmaceutical composition according to claim 47.

### Claim 51 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 42, 44, 45, 46, 47, 48, 49, 50)

An in vitro cell culture of haematopoietic cells according to any one of claims 1-14, or a granulocyte according to claim 42, or an in vitro cell culture of granulocytes according to any one of claims 44-46, or a pharmaceutical composition according to claim 47 for use, use or method according to any one of claims 48-50, wherein the cancer is a solid tumour cancer.

### Claim 52 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 42, 44, 45, 46, 47, 48, 49, 50)

An in vitro cell culture of haematopoietic cells according to any one of claims 1-14, or a granulocyte according to claim 42, or an in vitro cell culture of granulocytes according to any one of claims 44-46, or a pharmaceutical composition according to claim 47 for use, use or method according to any one of claims 48-50, wherein the cancer is one or more of: pancreatic cancer, liver cancer, oesophageal cancer, stomach cancer, cervical cancer, ovarian cancer, lung cancer, bladder cancer, kidney cancer, brain cancer, prostate cancer, myeloma cancer, non-Hodgkin's lymphoma (NHL), larynx cancer, uterine cancer, or breast cancer.

### Claim 53 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 42, 44, 45, 46, 47)

An in vitro method for selecting a subject for treatment with an in vitro cell culture of haematopoietic cells according to any one of claims 1-14, or a granulocyte according to claim 42, or an in vitro cell culture of granulocytes according to any one of claims 44-46, or a pharmaceutical composition according to claim 47, said method comprising:
a. admixing a granulocyte from said subject with a cancer cell line;
b. incubating said admixture;
c. measuring the % of cancer cells killed in said admixture; and
d. selecting a subject for treatment with an in vitro cell culture of haematopoietic cells according to any one of claims 1-14, or a granulocyte according to claim 42, or an in vitro cell culture of granulocytes according to any one of claims 44-46, or a pharmaceutical composition according to claim 47, when the granulocyte from said subject kills less than 70% of the cancer cells in the admixture.

### Claim 54 (depends on 53)

An in vitro method according to claim 53, wherein the subject is selected for treatment if the granulocyte from said subject kills less than 50% or less than 25% (preferably less than 10% or 5%) of the cancer cells in the admixture.

### Claim 55 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 42, 44, 45, 46, 47)

A cell bank comprising an in vitro cell culture of haematopoietic cells according to any one of claims 1-14, or a granulocyte according to claim 42, or an in vitro cell culture of granulocytes according to any one of claims 44-46, or a pharmaceutical composition according to claim 47.

### Claim 56 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 42, 44, 45, 46, 47)

A kit comprising:
a. in vitro cell culture of haematopoietic cells according to any one of claims 1-14, or a granulocyte according to claim 42, or an in vitro cell culture of granulocytes according to any one of claims 44-46, or a pharmaceutical composition according to claim 47; and
b. instructions for use of same in medicine.

### Claim 57 (depends on 56)

A kit according to claim 56, wherein said instructions are for use of same in treating cancer, preferably pancreatic cancer.