# US 12,215,350 B2 · Mononuclear cell derived NK cells

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 12,215,350 B2
- Document ID: US12215350B2
- Status: Granted
- Kind: B2
- Application number: 16/796,226
- Filed: 2020-02-20
- Published: 2021-01-14
- Granted: 2025-02-04
- Earliest claimed: 2019-07-08
- Assignees: ImmunityBio, Inc.
- Applicants: ImmunityBio, Inc.
- Inventors: Rohit Duggal; Ranjeet Sinha; Wenzhao Li; Jason Isaacson; Karl Marquez; Patrick Soon-Shiong
- Web page: https://patentgrep.com/patent/US12215350B2

## CPC

- C12N 5/0646: {Natural killers cells [NK], NKT cells}
- C12N 2500/30: Organic components
- C12N 2501/515: CD3, T-cell receptor complex
- C12N 2501/599: with CD designations not provided for elsewhere
- C12N 2501/998: Proteins not provided for elsewhere
- C12N 2506/11: from blood or immune system cells
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Cord blood or peripheral blood NK cells are prepared from whole blood mononuclear cells without the need to isolate CD34+ hematopoietic stem cells or NK cells, and without the need for a feeder layer. Advantageously, the methods presented herein use an enrichment process that uses antiCD16 agonist antibodies, antiCD3 antibodies, and N-803. Moreover, contemplated processes are suitable for adaptation into a fully automated production process (GMP in a box).

## Claims

### Claim 1

A method of expanding NK cells from a mixture of mononuclear cells, the method comprising:
providing in a container a mixture of the mononuclear cells that contains equal or less than 5% NK cells;
contacting the mixture of the mononuclear cells with an anti-CD16 antibody and N-803 to activate NK cells; and
feeding in the same container the activated NK cells with a medium containing N-803 until the activated NK cells constitute at least about 80% of all live cells or until the activated NK cells are enriched to an at least 80-fold expansion.

### Claim 2 (depends on 1)

The method of claim 1 wherein the mixture of the mononuclear cells is obtained from whole blood or cord blood.

### Claim 3 (depends on 1)

The method of claim 1, wherein the mixture of the mononuclear cells contains equal or less than 3% NK cells.

### Claim 4 (depends on 1)

The method of claim 1, wherein the anti-CD16 antibody is present at a concentration of between 0.05-0.5 mcg/ml.

### Claim 5 (depends on 1)

The method of claim 1, wherein the N-803 is present at a concentration of between 0.1-1.0 nM.

### Claim 6 (depends on 1)

The method of claim 1, wherein the step of contacting the mixture further includes contacting the mixture of the mononuclear cells with an anti-CD3 antibody.

### Claim 7 (depends on 6)

The method of claim 6, wherein the anti-CD3 antibody is present at a concentration of between 0.1-1.0 ng/ml.

### Claim 8 (depends on 1)

The method of claim 1, wherein the medium containing N-803 comprises human AB serum.

### Claim 9 (depends on 1)

The method of claim 1, wherein the step of feeding comprises sequentially feeding at an interval of about every 72 hours.

### Claim 10 (depends on 1)

The method of claim 1, wherein the step of feeding is performed until a total cell number of about 0.5-5.0×109 cells is reached.

### Claim 11 (depends on 1)

The method of claim 1, wherein the step of feeding the activated NK cells is performed until NK cells are enriched to an at least 100-fold expansion.

### Claim 12 (depends on 1)

The method of claim 1, wherein the step of feeding the activated NK cells is performed in an automated manner.

### Claim 13

A method of expanding NK cells in an automated bioreactor, the method comprising:
incubating a mixture of mononuclear cells in an activation medium containing N-803 and an anti-CD16 antibody for a time sufficient to activate NK cells;
wherein the mixture of mononuclear cells is contained in a cell culture container while incubating the mixture;
measuring growth of the cells while the cells are in the container; automatically feeding the cells using a medium containing N-803 until the activated NK cells constitute at least about 80% of all live cells or until the activated NK cells are enriched to an at least 80-fold expansion, wherein the feeding is controlled by a predetermined schedule and/or a result from the step of measuring growth of the cells;
terminating feeding the cells wherein the terminating is controlled by a predetermined schedule and/or a result from the step of measuring growth of the cells.

### Claim 14 (depends on 13)

The method of claim 13, wherein the container has a volume of between about 200 ml and about 2,500 ml.

### Claim 15 (depends on 13)

The method of claim 13, wherein the step of measuring growth of the cells is performed through a wall of the container.

### Claim 16 (depends on 13)

The method of claim 13, wherein the activation medium contains N-803 at a concentration of between 0.1-1.0 nM and the anti-CD16 antibody at a concentration of between 0.05-0.5 mcg/ml.

### Claim 17 (depends on 13)

The method of claim 13, wherein the time sufficient to activate NK cells is between 24 hours and 96 hours.

### Claim 18 (depends on 13)

The method of claim 13, wherein the medium containing N-803 contains N-803 at a concentration of between 0.1-1.0 nM.

### Claim 19 (depends on 13)

The method of claim 13, wherein the cells are fed until a total cell number of about 0.5-5.0×109 cells is reached.

### Claim 20 (depends on 13)

The method of claim 13, wherein the cells are fed until NK cells are enriched to an at least 100-fold expansion.