# US 11,180,731 B2 · Methods of treating acute myeloid leukemia and multiple myeloma using natural killer cells

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- Number: US 11,180,731 B2
- Document ID: US11180731B2
- Status: Granted
- Kind: B2
- Application number: 16/099,676
- Filed: 2017-05-05
- Published: 2019-05-23
- Granted: 2021-11-23
- Earliest claimed: 2016-05-07
- Assignees: Celularity Inc.
- Applicants: Celularity Inc.
- Inventors: Steven A. Fischkoff; Uri Herzberg; Lin Kang; Brian Murphy; Andrea Nordberg; Vanessa Voskinarian-Berse; Keith Wilson; Xiaokui Zhang; Han Myint; Mohamad Hussein; Robert J Hariri
- Web page: https://patentgrep.com/patent/US11180731B2

## CPC

- A61K 38/2013: {IL-2}
- A61K 40/15: Natural-killer [NK] cells; Natural-killer T [NKT] cells
- A61K 40/42: Cancer antigens
- A61P 35/00: Antineoplastic agents
- A61P 35/02: specific for leukemia
- C12N 5/0638: {Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]}
- C12N 5/0646: {Natural killers cells [NK], NKT cells}
- A61K 2239/31: characterized by the route of administration
- A61K 2239/38: characterised by the dose, timing or administration schedule
- A61K 2239/48: Blood cells, e.g. leukemia or lymphoma
- C12N 2501/145: Thrombopoietin [TPO]
- C12N 2501/22: Colony stimulating factors (G-CSF, GM-CSF)
- C12N 2501/2302: Interleukin-2 (IL-2)
- C12N 2501/2306: Interleukin-6 (IL-6)
- C12N 2501/2307: Interleukin-7 (IL-7)
- C12N 2501/2315: Interleukin-15 (IL-15)
- C12N 2501/26: Flt-3 ligand (CD135L, flk-2 ligand)
- C12N 2501/999: Small molecules not provided for elsewhere
- C12N 2506/11: from blood or immune system 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)

## Abstract

Provided herein are methods of treating acute myeloid leukemia (AML) and multiple myeloma (MM) by administering an effective amount of a cell population comprising natural killer cells, wherein the cell population comprising natural killer cells is produced by a three-stage method comprising culturing a population of hematopoietic stem or progenitor cells in media comprising stem cell mobilizing factors, e.g., three-stage methods of producing NK cells in media comprising stem cell mobilizing factors starting with hematopoietic stem or progenitor cells from cells of the placenta, for example, from placental perfusate (e.g., human placental perfusate) or other tissues, for example, umbilical cord blood or peripheral blood. Further provided herein are methods of using the NK cells produced by the three-stage methods provided herein to suppress the proliferation of acute myeloid leukemia cells. In certain embodiments, the NK cells produced by the three-stage methods described herein are used in combination with IL-2.

## Claims

### Claim 1

A method of treating an individual having acute myeloid leukemia by administering an effective amount of a cell population comprising natural killer cells, wherein the cell population comprising natural killer cells is produced by a method comprising the steps of:
(a) culturing hematopoietic stem or progenitor cells in a first medium comprising a stem cell mobilizing agent and thrombopoietin (Tpo) to produce a first population of cells;
(b) culturing the first population of cells in a second medium comprising a stem cell mobilizing agent and interleukin-15 (IL-15), and lacking Tpo, to produce a second population of cells; and
(c) culturing the second population of cells in a third medium comprising IL-2 and IL-15, and lacking a stem cell mobilizing agent and low-molecular weight heparin (LMWH), to produce a third population of cells;
wherein said stem cell mobilizing agent is an aryl hydrocarbon receptor inhibitor, wherein the third population of cells comprises natural killer cells that are CD56+, CD3−, CD16− or CD16÷, and CD94÷ or CD94-, wherein at least 80% of the natural killer cells are viable; and
wherein the method of treatment additionally comprises administering to the patient an effective amount of IL-2.

### Claim 2 (depends on 1)

The method of claim 1, wherein said hematopoietic stem cells are CD34+ hematopoietic stem cells, or wherein said hematopoietic stem cells are placental hematopoietic stem cells.

### Claim 3 (depends on 2)

The method of claim 2, wherein said placental hematopoietic stem cells are obtained from, or obtainable from, human placental perfusate, or wherein said placental hematopoietic stem cells are obtained from, or obtainable from, nucleated cells isolated from human placental perfusate.

### Claim 4 (depends on 1)

The method of claim 1, wherein said aryl hydrocarbon receptor inhibitor is StemRegenin-1 (SR-1) (4-(2-(2-(benzo[b]thiophen-3-yl)-9-isopropyl-9H-purin-6-ylamino)ethyl)phenol), wherein said aryl hydrocarbon receptor inhibitor is resveratrol, wherein said aryl hydrocarbon receptor inhibitor is the compound CH223191 (1-Methyl-N-[2-methyl-4-[2-(2-methylphenyl)diazenyl]phenyl-1H-pyrazole-5-carboxamide].

### Claim 5 (depends on 1)

The method of claim 1, wherein said first medium additionally comprises one or more of Low Molecular Weight Heparin (LMWH), Flt-3 Ligand (Flt-3L), stem cell factor (SCF), IL-6, IL-7, granulocyte colony-stimulating factor (G-CSF), or granulocyte-macrophage-stimulating factor (GM-CSF), or wherein said first medium comprises each of LMWH, Flt-3L, SCF, IL-6, IL-7, G-CSF, and GM-CSF.

### Claim 6 (depends on 5)

The method of claim 5, wherein said LMWH, Flt-3, SCF, IL-6, IL-7, G-CSF, and/or GM-CSF are not comprised within an undefined component of the first medium, second medium or third medium, or wherein said LMWH, Flt-3, SCF, IL-6, IL-7, G-CSF, and/or GM-CSF are not comprised within serum.

### Claim 7 (depends on 1)

The method of claim 1, wherein said second medium additionally comprises one or more of LMWH, Flt-3, SCF, IL-6, IL-7, G-CSF, and GM-CSF, or wherein said second medium additionally comprises each of LMWH, Flt-3, SCF, IL-6, IL-7, G-CSF, and GM-CSF.

### Claim 8 (depends on 1)

The method of claim 1, wherein said third medium additionally comprises one or more of SCF, IL-6, IL-7, G-CSF, or GM-CSF, or wherein said third medium comprises each of SCF, IL-6, G-CSF, and GM-CSF.

### Claim 9 (depends on 1)

The method of claim 1, wherein any of said first medium, second medium or third medium comprises human serum-AB, wherein any of said first medium, second medium or third medium comprises 2-mercaptoethanol, or wherein any of said first medium, second medium or third medium comprises gentamycin.

### Claim 10 (depends on 1)

The method of claim 1, wherein said method comprises culturing the hematopoietic stem cells in the first medium for 7-13 days, wherein said method comprises culturing the hematopoietic stem cells in the first medium for 8-12 days, or wherein said method comprises culturing the hematopoietic stem cells in the first medium for about 10 days.

### Claim 11 (depends on 1)

The method of claim 1, wherein said method comprises culturing said first population of cells in said second medium for 2-6 days, wherein said method comprises culturing said first population of cells in said second medium for 3-5 days, or wherein said method comprises culturing said first population of cells in said second medium for about 4 days.

### Claim 12 (depends on 1)

The method of claim 1, wherein said method comprises culturing said second population of cells in said third medium for 10-30 days, wherein said method comprises culturing said second population of cells in said third medium for 15-25 days, or wherein said method comprises culturing said second population of cells in said third medium for about 21 days.

### Claim 13 (depends on 1)

The method of claim 1, wherein said method produces natural killer cells that comprise at least 20% CD56+CD3− natural killer cells, wherein said method produces natural killer cells that comprise at least 40% CD56+CD3− natural killer cells, wherein said method produces natural killer cells that comprise at least 60% CD56+CD3−natural killer cells, or wherein said method produces natural killer cells that comprise at least 80% CD56+CD3− natural killer cells.

### Claim 14 (depends on 1)

The method of claim 1, additionally comprising cryopreserving said population of cells after step (c), additionally comprising cryopreserving said natural killer cells after step (c), or wherein said natural killer cells have been cryopreserved prior to said contacting or said administering.

### Claim 15 (depends on 1)

The method of claim 1, wherein said individual is administered about 1×106, 3×106, 10×106, or 30×106 natural killer cells per kilogram of the individual.

### Claim 16

A method of treating an individual having multiple myeloma by administering an effective amount of a cell population comprising natural killer cells, wherein the cell population comprising natural killer cells is produced by a method comprising the steps of:
(a) culturing hematopoietic stem or progenitor cells in a first medium comprising a stem cell mobilizing agent and thrombopoietin (Tpo) to produce a first population of cells;
(b) culturing the first population of cells in a second medium comprising a stem cell mobilizing agent and interleukin-15 (IL-15), and lacking Tpo, to produce a second population of cells; and
(c) culturing the second population of cells in a third medium comprising IL-2 and IL-15, and lacking a stem cell mobilizing agent and LMWH, to produce a third population of cells;
wherein said stem cell mobilizing agent is an aryl hydrocarbon receptor inhibitor, wherein the third population of cells comprises natural killer cells that are CD56±, CD3−, CD16− or CD16÷, and CD94÷ or CD94−, wherein at least 80% of the natural killer cells are viable; and
wherein the method of treatment additionally comprises administering to the patient an effective amount of IL-2.

### Claim 17

A method of suppressing the proliferation of multiple myeloma cells comprising contacting the multiple myeloma cells with a plurality of natural killer cells, wherein the natural killer cells are by a method comprising the steps of:
(a) culturing hematopoietic stem or progenitor cells in a first medium comprising a stem cell mobilizing agent and thrombopoietin (Tpo) to produce a first population of cells;
(b) culturing the first population of cells in a second medium comprising a stem cell mobilizing agent and interleukin-15 (IL-15); and lacking Tpo, to produce a second population of cells; and
(c) culturing the second population of cells in a third medium comprising IL-2 and IL-15, and lacking a stem cell mobilizing agent and LMWH, to produce a third population of cells;
wherein said stein cell mobilizing agent is an aryl hydrocarbon receptor inhibitor, wherein the third population of cells comprises natural killer cells that are CD56+, CD3—, CD16− or CD16÷, and CD94÷ or CD94−, wherein at least 80% of the natural killer cells are viable; and
wherein said method of suppressing additionally comprises contacting the multiple myeloma cells with IL-2.

### Claim 18 (depends on 17)

The method of claim 17, wherein said natural killer cells have been cryopreserved prior to said contacting or said administering.