# US 12,180,492 B2 · Methods and systems for producing a protein of interest in a plant

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- Number: US 12,180,492 B2
- Document ID: US12180492B2
- Status: Granted
- Kind: B2
- Application number: 17/750,768
- Filed: 2022-05-23
- Published: 2022-12-15
- Granted: 2024-12-31
- Earliest claimed: 2021-06-09
- Assignees: ImmunityBio, Inc.
- Applicants: ImmunityBio, Inc.
- Inventors: Patrick Soon-Shiong
- Web page: https://patentgrep.com/patent/US12180492B2

## CPC

- C12N 15/8257: {for the production of primary gene products, e.g. pharmaceutical products, interferon}
- C12N 15/8258: {for the production of oral vaccines (antigens) or immunoglobulins}
- A01H 3/02: by controlling duration, wavelength, intensity, or periodicity of illumination
- A01H 3/04: by treatment with chemicals
- C07K 14/5443: {IL-15}
- C07K 14/7155: {for interleukins [IL]}
- C07K 16/46: Hybrid immunoglobulins (hybrids of an immunoglobulin with a peptide not being an immunoglobulin C07K19/00)
- C12N 15/8251: {Amino acid content, e.g. synthetic storage proteins, altering amino acid biosynthesis}
- C07K 2317/52: Constant or Fc region; Isotype
- C07K 2319/30: Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
- C07K 2319/32: fusions with soluble part of a cell surface receptor, "decoy receptors"
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A01H: NEW PLANTS OR {NON-TRANSGENIC} PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- 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)

## Abstract

Methods and systems for producing a protein of interest within a plant or a portion of a plant are provided herein. The method includes introducing one or more nucleic acid into the plant or the portion of the plant, the nucleic acid including a nucleotide sequence encoding the protein of interest and incubating the plant or the portion of the plant under conditions that permit the expression of the nucleotide sequence encoding the protein of interest. The method also includes adding a medium amendment including a calcium carbonate source, such as aragonite, to a medium environment of the plant.

## Claims

### Claim 1

A method for producing a protein of interest within a plant or a portion of a plant comprising:
a) introducing one or more nucleic acids into the plant or the portion of the plant, the nucleic acid comprising a nucleotide sequence encoding the protein of interest and operatively linked to a regulatory region;
b) incubating the plant or the portion of the plant under conditions that permit expression of the nucleotide sequence encoding the protein of interest, thereby producing the protein of interest; and
c) adding a medium amendment to a medium environment of the plant, wherein the medium amendment comprises a calcium carbonate source comprising aragonite,
wherein the protein of interest is nogapendekin-alfa-inbakicept (NAI), a heterodimeric bifunctional fusion complex comprising a soluble TGF-BRII domain and an IL-15/IL15RαSu domain linked by a tissue factor linker, a T×M, or a mAb; and
wherein the plant is from a genus selected from the group consisting of Arabidopsis, Nicotiana, Brassica, Ipomoea, Zea, Sorghum, Carthamus, Glycine, Triticum, Solanum, Avena, Secale, Medicago, Helianthus, Gossypium, Hordeum, Oryza, Panax, and Pisum.

### Claim 2 (depends on 1)

The method of claim 1, wherein the heterodimeric bifunctional fusion complex comprising a soluble TGF-βRII domain and an IL-15/IL15RαSu domain linked by a tissue factor linker is HCW9218 or HCW9228.

### Claim 3 (depends on 1)

The method of claim 1, wherein the T×M is a complex comprising an IL-15N72D:IL-15RαSu/Fc scaffold linked to a binding domain.

### Claim 4 (depends on 3)

The method of claim 3, wherein the binding domain recognizes an immune checkpoint molecule, immune signaling molecule, or a disease antigen.

### Claim 5 (depends on 1)

The method of claim 1, wherein in step b), the nucleic acid is transiently expressed in the plant.

### Claim 6 (depends on 1)

The method of claim 1, wherein in step b), the nucleic acid is stably expressed in the plant.

### Claim 7 (depends on 1)

The method of claim 1, wherein the medium environment comprises soil.

### Claim 8 (depends on 1)

The method of claim 1, wherein the aragonite is in the form of particles having a particle size of about 3 μm to about 10 μm in diameter.

### Claim 9 (depends on 1)

The method of claim 1, wherein the aragonite is oolitic aragonite.

### Claim 10 (depends on 9)

The method of claim 9, wherein the oolitic aragonite is unprocessed.

### Claim 11 (depends on 1)

The method of claim 1, wherein the calcium carbonate source is present in the medium amendment in an amount of at least about 10 wt %, based on total weight of the medium amendment.

### Claim 12 (depends on 1)

The method of claim 1, wherein the medium amendment further comprises one or more of: a binding agent, an organic material, compost, gypsum, borax, weathered lignite, phosphate, paramagnetic basalt powder, glacial gravel dust, potassium, fulvic acid powder, humic acid powder, a filler, an herbicide, a fungicide, fungus, and a bacteria.

### Claim 13 (depends on 1)

The method of claim 1, wherein the medium amendment is added to the medium environment prior to seeding, during seeding, after seeding, or a combination thereof.

### Claim 14 (depends on 1)

The method of claim 1, further comprising a step d) of harvesting the plant or the portion of the plant.

### Claim 15 (depends on 14)

The method of claim 14, wherein the step d) further comprises purifying the protein of interest.

### Claim 16 (depends on 1)

The method of claim 1, wherein incubating the plant includes at least one of:
modifying a duration of light supplied to the plant during one or more specified daily time periods, during growth of the plant;
modifying an intensity of light supplied to the plant during growth of the plant; and
modifying a wavelength of light supplied to the plant during growth of the plant.

### Claim 17 (depends on 16)

The method of claim 16, further comprising determining a level of production of the protein of interest, wherein modifying the duration, intensity and/or wavelength of light includes modifying the duration, intensity, and/or wavelength of light according to the determined level of production of the protein of interest.

### Claim 18 (depends on 16)

The method of claim 16, further comprising determining a level of growth of the plant, wherein modifying the duration, intensity, and/or wavelength of light includes modifying the duration, intensity and/or wavelength of light according to the determined level of growth of the plant.

### Claim 19 (depends on 17)

The method of claim 17, wherein:
determining a level of growth of the plant and/or the production of the protein of interest includes determining the level of growth and/or production of the protein of interest using at least one sensor associated with the plant; and
the at least one sensor includes at least one of a dendrometer, a strain sensor, a light sensor, a temperature sensor, a moisture sensor, and a camera.

### Claim 20 (depends on 1)

The method of claim 1, wherein the plant is from a Nicotiana genus.