# US 2020/0399596 A1 · B-cell cultivation method

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- Number: US 2020/0399596 A1
- Document ID: US20200399596A1
- Status: Published application
- Kind: A1
- Application number: 16/888,213
- Filed: 2020-05-29
- Published: 2020-12-24
- Earliest claimed: 2017-11-30
- Assignees: Hoffmann-La Roche Inc.
- Applicants: Hoffmann-La Roche Inc.
- Inventors: Sonja OFFNER; Friederike JUNG
- Web page: https://patentgrep.com/patent/US20200399596A1

## CPC

- C12N 5/0635: {B lymphocytes}
- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C12N 2500/30: Organic components
- C12N 2501/2301: Interleukin-1 (IL-1)
- C12N 2501/2302: Interleukin-2 (IL-2)
- C12N 2501/2304: Interleukin-4 (IL-4)
- C12N 2501/2306: Interleukin-6 (IL-6)
- C12N 2501/231: Interleukin-10 (IL-10)
- C12N 2501/2321: Interleukin-21 (IL-21)
- C12N 2501/25: Tumour necrosing factors [TNF]
- C12N 2502/1107: B cells
- C12N 2502/1114: T cells
- C12N 2502/1157: Monocytes, macrophages
- C12N 2502/1185: Thymus cells
- C12N 2529/00: Culture process characterised by the use of electromagnetic stimulation
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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

Herein is reported a method for the co-cultivation of single deposited B-cells, which can be of any source, with EL-4 B5 feeder cells in a suitable co-cultivation medium. In the herein reported methods the EL-4 B5 cells have been irradiated with a dose of less than 40 Gy, preferably 9.5 Gy or less. Thereby the EL-4 B5 cells have a higher viability and maintain the ability to divide in cultivation at doses less than 6 Gy.

## Claims

### Claim 1

A method for cultivating one or more B-cells comprising the step of
co-cultivating one or more B-cells with EL-4 B5 cells,
whereby the EL-4 B5 cells have been irradiated prior to the co-cultivation with a dose of 9.5 Gy or less, and
whereby the number of EL-4 B5 cells at the start of the co-cultivating is less than 5×104 per B-cell.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the co-cultivating is additionally in the presence of a feeder mix.

### Claim 3 (depends on 2)

The method according to claim 2, wherein the feeder mix comprises one or more of
i) interleukin-1 beta and tumor necrosis factor alpha,
ii) interleukin-2 (IL-2) and/or interleukin-10 (IL-10),
iii) Staphylococcus aureus strain Cowan's cells (SAC),
iv) interleukin-21 (IL-21) and optionally interleukin-2 (IL-2),
v) B-cell activation factor of the tumor necrosis factor family (BAFF),
vi) interleukin-6 (IL-6),
vii) interleukin-4 (IL-4), and
viii) thymocyte cultivation supernatant.

### Claim 4 (depends on 3)

The method according to claim 3, wherein the feeder mix comprises
about 2 ng/ml (murine) IL-1beta,
about 2 ng/ml (murine) TNFalpha,
about 50 ng/ml (murine) IL-2,
about 10 ng/ml (murine) IL-10, and
about 10 ng/ml (murine) IL-6,
or a fraction thereof.

### Claim 5 (depends on 4)

The method according to claim 4, wherein the fraction of the feeder mix is in the range of from 1.0- to 0.015-times of each of said concentrations of IL-1beta, TNFalpha, IL-2, IL-10, and IL-6.

### Claim 6 (depends on 3)

The method according to claim 3, wherein the feeder mix further comprises phorbol myristate acetate.

### Claim 7 (depends on 2)

The method according claim 2, wherein the method is for the cultivation of a single deposited B-cell.

### Claim 8 (depends on 7)

The method according to claim 7, wherein the co-cultivating is for 5 to 10 days.

### Claim 9 (depends on 1)

The method according to claim 1, wherein the co-cultivating of the one or more B-cells is with about 10,000 to about 30,000 EL-4 B5 cells, which have been irradiated with gamma radiation of a dose in the range of about 3 Gy to about 6 Gy.

### Claim 10 (depends on 1)

The method according to claim 1, wherein the co-cultivating of the one or more B-cells is with about 10,000 to about 30,000 EL-4 B5 cells, which have been irradiated with gamma radiation of the dose in the range of about 3 Gy to about 6 Gy, wherein the feeder mix comprises about 0.06 ng/ml IL-1beta, about 0.06 ng/ml TNFalpha, about 1.5 ng/ml IL-2, about 0.3 ng/ml IL-10, about 0.3 ng/ml IL-6, and about 0.25 ng/ml-0.5 ng/ml PMA.

### Claim 11 (depends on 2)

The method according to claim 2, wherein the co-cultivating of the one or more B-cells is with about 2,500 to about 7,500 EL-4 B5 cells, which have been irradiated with gamma radiation of a dose in the range of 0 Gy to less than 3 Gy.

### Claim 12 (depends on 1)

The method according to claim 1, wherein the co-cultivating of the one or more B-cells is with about 2,500 to about 7,500 EL-4 B5 cells, which have been irradiated with gamma radiation of the dose in the range of 0 Gy to less than 3 Gy, wherein the feeder mix comprises about 0.06 ng/ml to about 0.2 ng/ml IL-1beta, about 0.06 ng/ml to about 0.2 ng/ml TNFalpha, about 1.5 ng/ml to about 5 ng/ml IL-2, about 0.3 ng/ml to about 1 ng/ml IL-10, about 0.3 ng/ml to about 1 ng/ml IL-6, and about 0.43 ng/ml-0.73 ng/ml PMA.

### Claim 13 (depends on 5)

The method according to claim 5, wherein the feeder mix further comprises about 0.125 ng/ml-1 ng/ml phorbol myristate acetate

### Claim 14 (depends on 13)

The method according to any one of claim 13, wherein the method is for the cultivation of a single deposited B-cell.

### Claim 15 (depends on 14)

The method according to claim 14, wherein the co-cultivating is for 5 to 10 days.

### Claim 16 (depends on 8)

The method according to claim 8, wherein the co-cultivating of the one or more B-cells is with about 10,000 to about 30,000 EL-4 B5 cells, which have been irradiated with gamma radiation of a dose in the range of about 3 Gy to about 6 Gy

### Claim 17 (depends on 16)

The method according to claim 16, further wherein the feeder mix comprises about 0.06 ng/ml IL-1beta, about 0.06 ng/ml TNFalpha, about 1.5 ng/ml IL-2, about 0.3 ng/ml IL-10, about 0.3 ng/ml IL-6, and about 0.25 ng/ml-0.5 ng/ml PMA

### Claim 18 (depends on 15)

The method according to claim 15, wherein the co-cultivating of the one or more B-cells is with about 2,500 to about 7,500 EL-4 B5 cells, which have been irradiated with gamma radiation of a dose in the range of 0 Gy to less than 3 Gy.

### Claim 19 (depends on 11)

The method according to claim 11, wherein the feeder mix comprises about 0.06 ng/ml to about 0.2 ng/ml IL-1beta, about 0.06 ng/ml to about 0.2 ng/ml TNFalpha, about 1.5 ng/ml to about 5 ng/ml IL-2, about 0.3 ng/ml to about 1 ng/ml IL-10, about 0.3 ng/ml to about 1 ng/ml IL-6, and about 0.43 ng/ml-0.73 ng/ml PMA.

### Claim 20 (depends on 15)

The method according to claim 15, wherein the co-cultivating of the one or more B-cells is with about 2,500 to about 7,500 EL-4 B5 cells, which have been irradiated with gamma radiation of the dose in the range of 0 Gy to less than 3 Gy, wherein the feeder mix comprises about 0.06 ng/ml to about 0.2 ng/ml IL-1beta, about 0.06 ng/ml to about 0.2 ng/ml TNFalpha, about 1.5 ng/ml to about 5 ng/ml IL-2, about 0.3 ng/ml to about 1 ng/ml IL-10, about 0.3 ng/ml to about 1 ng/ml IL-6, and about 0.43 ng/ml-0.73 ng/ml PMA