Traitement en cours

Veuillez attendre...

Paramétrages

Paramétrages

Aller à Demande

1. WO2020109953 - LYMPHOCYTES T GAMMA DELTA ET LEURS UTILISATIONS

Note: Texte fondé sur des processus automatiques de reconnaissance optique de caractères. Seule la version PDF a une valeur juridique

[ EN ]

CLAIMS

1. A method of expanding and isolating g d T cells from human peripheral blood mononuclear cells (PBMCs), the method comprising:

a. obtaining human PBMCs;

b. culturing the human PBMCs in a culture media comprising zoledronic acid, interleukin-2 (IL-2), and interleukin-15 (IL-15) to expand the g d T cell; and

c. isolating the g d T cells.

2. The method of claim 1, wherein the concentration of the zoledronic acid is about 1 mM to about 20 mM.

3. The method of claim 2, wherein the concentration of the zoledronic acid is about 5 mM.

4. The method of any one of clams 1-3, wherein the concentration of the IL-2 is about 50 IU/mL to about 5000 IU/mL.

5. The method of claim 4, wherein the concentration of IL-2 is about 100 IU/mL to about 1000 IU/mL.

6. The method of any one of claims 1-5, wherein the IL-2 is recombinant human IL-2 (rhIL-2).

7. The method of any one of claims 1-6, wherein the concentration of IL-15 is about 1 ng/mL to about 100 ng/mL.

8. The method of claim 7, wherein the concentration of IL-15 is about 10 ng/mL. 9. The method of any one of claims 1-8, wherein the IL-15 is recombinant human IL-15 (rhIL-15).

10. The method of any one of claims 1-9, wherein the g d T cell is a V g9V d2 T cell. 11. The method of any one of claims 1-10, wherein the g d T cells are isolated by flow cytometry, magnetic separation, and negative selection.

12. An isolated g d T cell produced by the method of claim 11.

13. A method of generating a chimeric antigen receptor (CAR)- g d T cell, the method comprising:

a. obtaining an isolated g d T cell of claim 12;

b. contacting the g d T cell with a nucleic acid encoding a chimeric antigen receptor (CAR), the CAR comprising:

i. an extracellular domain;

ii. a transmembrane domain; and

iii. an intracellular signaling domain,

wherein the CAR optionally further comprises a signal peptide at the amino terminus and a hinge region connecting the extracellular domain and the transmembrane domain, and wherein contacting the g d T cell with the nucleic acid encoding the CAR generates a CAR g d T cell.

14. The method of claim 13, wherein the CAR comprises:

i. an extracellular domain comprising an antigen binding domain and/or an antigen binding fragment;

ii. a transmembrane domain comprising a CD8 a transmembrane domain; iii. an intracellular signaling domain comprising a CD3 z or 4-1BB

intracellular domain;

iv. a signal peptide comprising a CD8 a signal peptide; and

v. a hinge region comprising a CD8 a hinge region.

15. The method of claim 14, wherein the CAR comprises:

i. the transmembrane domain having an amino acid sequence at least 90% identical to SEQ ID NO:1;

ii. the intracellular domain having an amino acid sequence at least 90%

identical to SEQ ID NO:2 or SEQ ID NO:3;

iii. the signal peptide having an amino acid sequence at least 90% identical to SEQ ID NO:4; and

iv. the hinge region having an amino acid sequence at least 90% identical to SEQ ID NO:5.

16. The method of claim 14 or 15, wherein the extracellular domain comprises an antigen binding domain and/or an antigen binding fragment that specifically binds a tumor antigen.

17. The method of any one of claims 13-16, wherein the CAR comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:22-29.

18. A CAR- g d T cell produced by the method of any one of claims 13-17.

19. A pharmaceutical composition comprising the CAR- g d T cell of claim 18 and a pharmaceutically acceptable carrier.

20. A method of treating or preventing a disease or condition in a subject in need thereof, the method comprising administering a therapeutically effective amount of the pharmaceutical composition of claim 19.

21. The method of claim 20, wherein the disease or condition is cancer.

22. The method of claim 21, wherein the cancer is selected from a solid cancer or a liquid cancer.

23. The method of claim 22, wherein the cancer is selected from the group consisting of a lung cancer, a gastric cancer, a colon cancer, a hepatocellular carcinoma, a renal cell carcinoma, a bladder urothelial carcinoma, a metastatic melanoma, a breast cancer, an ovarian cancer, a cervical cancer, a head and neck cancer, a pancreatic cancer, an endometrial cancer, a prostate cancer, a thyroid cancer, a glioma, a glioblastoma, and other solid tumors, and a non-Hodgkin’s lymphoma (NHL), a Hodgkin’s

lymphoma/disease (HD), an acute lymphocytic leukemia (ALL), a chronic lymphocytic leukemia (CLL), a chronic myelogenous leukemia (CML), a multiple myeloma (MM), an acute myeloid leukemia (AML), and other liquid tumors.

24. The method of claim 20, wherein the disease or condition is an autoimmune disease.

25. The method of claim 24, wherein the autoimmune disease is selected from the group consisting of alopecia, amyloidosis, ankylosing spondylitis, Castleman disease (CD), celiac disease, crohn’s disease, endometriosis, fibromyalgia, glomerulonephritis, Graves’ disease, Guillain-Barre syndrome, IgA nephropathy, lupus, lyme disease, Meniere;s disease, multiple sclerosis, narcolepsy, neutropenia, psoriasis, psoriatic arthritis, rheumatoid arthritis, sarcoidosis, scleroderma, type 1 diabetes, ulcerative colitis, and vitiligo.

26. A method of producing a pharmaceutical composition comprising a CAR- g d T cell, wherein the methods comprises combining the CAR- g d T cell of claim 18 with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.