# US 12,600,945 B2 · Human Vγ9Vδ2T cell proliferation culture method and culture medium

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- Number: US 12,600,945 B2
- Document ID: US12600945B2
- Status: Granted
- Kind: B2
- Application number: 17/418,103
- Filed: 2019-02-19
- Published: 2021-10-28
- Granted: 2026-04-14
- Earliest claimed: 2018-12-24
- Assignees: Guangdong Gd Kongming Biotech LLC
- Applicants: Guangdong Gd Kongming Biotech LLC
- Inventors: Zhinan Yin; Yangzhe Wu; Yan Xu
- Web page: https://patentgrep.com/patent/US12600945B2

## CPC

- C12N 5/0636: {T lymphocytes}
- A61K 40/11: T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
- A61K 40/42: Cancer antigens
- A61P 35/00: Antineoplastic agents
- A61K 2239/31: characterized by the route of administration
- A61K 2239/38: characterised by the dose, timing or administration schedule
- A61K 2239/55: Lung
- C12N 2500/38: Vitamins
- C12N 2501/2302: Interleukin-2 (IL-2)
- C12N 2501/2315: Interleukin-15 (IL-15)
- C12N 2501/999: Small molecules not provided for elsewhere
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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

## Abstract

A human Vγ9Vδ2T cell proliferation method and a culture medium. The method comprises: first stimulating a human Vγ9Vδ2T cell with a culture medium containing interleukin-2 and phosphonic acid compounds, and then culturing the human Vγ9Vδ2T cell with a culture medium added with interleukin-15 and vitamin C to achieve proliferation culture. The culture medium additionally comprises interleukin-15 and vitamin C. Compared with a conventional proliferation method and a conventional culture medium, the method and the culture medium can improve the proliferation efficiency and cell purity of the human Vγ9Vδ2T cell. The human Vγ9Vδ2T cell obtained by culture in the method has stronger anti-apoptotic ability and longer cell survival time, and moreover, the expression level of critical killer molecules NKG2D thereof is higher, thereby having stronger killing ability to tumor cells.

## Claims

### Claim 1

A method for selectively proliferating Vγ9Vδ2T cells, comprising:
step A, culturing a composition containing the T cells, by using a first culture medium which is able to stimulate T cells, so as to stimulate the T cells, wherein the first culture medium comprises interleukin-2 and phosphonic acid compounds, and the phosphonic acid compounds are selected from the group consisting of zoledronic acid, etidronic acid, ibandronic acid, pamidronic acid, alendronic acid, risedronic acid, minodronic acid and combinations thereof; and
step B, using a second culture medium to culture the stimulated T cells, wherein the second culture medium contains interleukin-2, interleukin-15, and vitamin C or a derivative of vitamin C, thereby selectively proliferating the Vy9V82T cells.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the first culture medium comprises interleukin-15, and vitamin C or a derivative of vitamin C.

### Claim 3 (depends on 1)

The method according to claim 1, wherein the derivative of vitamin C is selected from the group consisting of vitamin C ethyl ether, vitamin C palmitate, vitamin C glucoside, vitamin C magnesium phosphate and vitamin C sodium phosphate and combinations thereof.

### Claim 4 (depends on 1)

The method according to claim 1, wherein in the second culture medium, a concentration of the interleukin-15 is 1-1000 ng/ml.

### Claim 5 (depends on 1)

The method according to claim 1, wherein in the second culture medium, a concentration of the vitamin C or the derivative of vitamin C is 10 μM-800 mM.

### Claim 6 (depends on 1)

The method according to claim 1, wherein in the second culture medium, a concentration of the interleukin-2 is 1-1000 ng/ml.

### Claim 7 (depends on 1)

The method according to claim 1, wherein the composition containing the T cells comprises peripheral blood mononuclear cells extracted from peripheral blood of human body.

### Claim 8 (depends on 1)

The method according to claim 1, wherein the step A comprises culturing the composition containing the T cells for 60-100 hours.

### Claim 9 (depends on 1)

The method according to claim 1, wherein the second culture medium further comprises a basic culture medium, wherein the basic culture medium is selected from the group consisting of Roswell Park Memorial Institute Medium 1640, Dulbecco's Modified Eagle Medium, Minimum Essential Medium, Roswell Park Memorial Institute Medium, Opti-Minimum Essential Medium and combinations thereof.

### Claim 10 (depends on 1)

A method for treating a malignant disease, comprising:
(1) extracting peripheral blood mononuclear cells from peripheral blood of a subject in need;
(2) proliferating the peripheral blood mononuclear cells through the method according to claim 1; and
(3) administering proliferated product to the subject in need,
wherein the peripheral blood mononuclear cells are used as the composition containing the T cells, and the malignant disease is lung cancer.