Method for preparing antigen-specific cytotoxic T-cells by using activated B-cells and use thereof

• US 10,443,040 B2
• Filed: 09/29/2017
• Issued: 10/15/2019
• Est. Priority Date: 03/31/2015
• Status: Active Grant

• Current Assignee: Seoul National University R&DB Foundation (Seoul National University)
• Original Assignee: Seoul National University R&DB Foundation (Seoul National University)
• Inventors: Kang, Hyoung Jin, Yun, Sun Ok, Kang, Chang-Yuil
• Primary Examiner(s): Gamett, Daniel C
• Application Number:
  US15/720,508
• Publication Number:
  US 20180023054A1
• Time in Patent Office: 746 Days
• Field of Search: None
• CPC Class Codes: A61K 2039/5156 expressing foreign proteins
• CPC Class Codes: A61K 2039/5158 Antigen-pulsed cells, e.g. T-cells
• CPC Class Codes: A61K 35/17 Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes when activated by a specific antigen A61K39/00
• CPC Class Codes: A61K 39/0011 Cancer antigens
• CPC Class Codes: A61K 39/001153 Wilms tumor 1 [WT1]
• CPC Class Codes: A61K 39/001156 Tyrosinase and tyrosinase related proteinases [TRP-1 or TRP-2]
• CPC Class Codes: A61K 39/001157 Telomerase or TERT [telomerase reverse transcriptase]
• CPC Class Codes: A61K 39/001181 Alpha-feto protein
• CPC Class Codes: A61K 39/001182 Carcinoembryonic antigen [CEA]
• CPC Class Codes: A61K 39/001186 MAGE
• CPC Class Codes: A61K 39/001197 Breakpoint cluster region-abelson tyrosine kinase [BCR-ABL]
• CPC Class Codes: A61K 39/235 Adenoviridae
• CPC Class Codes: A61K 39/245 Herpetoviridae, e.g. herpes simplex virus
• CPC Class Codes: A61K 39/4612 B-cells
• CPC Class Codes: A61K 39/4622 Antigen presenting cells
• CPC Class Codes: A61K 39/464453 Wilms tumor 1 [WT1]
• CPC Class Codes: A61K 39/464838 Viral antigens
• CPC Class Codes: C07K 14/52 Cytokines; Lymphokines; Interferons
• CPC Class Codes: C12N 2501/05 Adjuvants
• CPC Class Codes: C12N 2501/2304 Interleukin-4 (IL-4)
• CPC Class Codes: C12N 2501/2307 : Interleukin-7 (IL-7)
• CPC Class Codes: C12N 2501/2315 : Interleukin-15 (IL-15)
• CPC Class Codes: C12N 2502/1107 : B cells
• CPC Class Codes: C12N 2502/99 : genetically modified cells
• CPC Class Codes: C12N 2710/10034 : Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
• CPC Class Codes: C12N 2710/16134 : Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
• CPC Class Codes: C12N 2710/16234 : Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
• CPC Class Codes: C12N 5/0638 : Cytotoxic T lymphocytes [CTL] or lymphokine activated killer cells [LAK]

Abstract

Disclosed in the present application are: a method for preparing in vitro/ex vivo antigen-specific cytotoxic T-cells by using B cells treated with biological response modifier; and a use thereof. The cytotoxic T-cells prepared by the method of the present application can be used advantageously for treating infectious disease and cancer and the like.

Claims

1. A method of producing antigen-specific cytotoxic T cells, comprising the steps of:

providing human peripheral blood mononuclear cells;

separating the peripheral blood mononuclear cells into B cells and other mononuclear cells;

treating the B cells with any one of biological response modifiers selected from the group consisting of alpha-galactosylceramide, alpha-glucuronosylceramide, phosphatidylinositol tetramannoside, isoglobotrihexylceramide, ganglioside GD3, phosphatidylcholine, beta-galactosylceramide, lipophosphoglycan, glycoinositol phospholipid, alpha-galactosylceramide analogs, including beta-anomer galactosylceramide and alpha-anomer galactosylceramide to produce activated B cells;

introducing into the activated B cells an antigen polypeptide or a DNA encoding an antigen to produce activated B cells loaded with an antigen;

mixing a first batch of the antigen-loaded activated B cells and the other mononuclear cells separated from the peripheral blood mononuclear cells to produce a first mixture of the cells;

subjecting the first mixture of the cells to a first incubation;

adding a second batch of the antigen-loaded activated B cells to the first mixture of the cells after the first incubation to produce a second mixture; and

subjecting the second mixture to a second incubation,

wherein a cytokine is added only during the second incubation step and wherein the antigen-specific cytotoxic T cells are generated during the first and the second incubation steps.

2. The method of claim 1, wherein the cytokine is at least one of IL-15, IL-2, IL-4, IL-7 and IL-9.

3. The method of claim 2, wherein the cytokine is added at a concentration of 10-100 ng/ml.

4. The method of claim 1, wherein the antigen is an antigen from a pathogen including a virus, a pathogenic bacterium or a parasite, or a tumor antigen.

5. The method of claim 4, wherein the antigen from the virus is selected from the group consisting of an Epstein-Barr virus (EBV) antigen including E1dGALMP2 or BZLF1, a cytomegalovirus (CMV) antigen including pp65 or 1E1, an Adenovirus (Adv) antigen including HEXON or PENTON, an influenza virus antigen, a human papilloma virus (HPV) antigen, a vesicular stomatitis virus antigen, a hepatitis A, B, C, D or G virus antigen including a core antigen or a surface antigen, a respiratory synctytial virus antigen, a herpes simplex virus antigen, a human immunodeficiency virus antigen including GP-120, GP-160, p18, Tat, Gag, Pol or Env, and combinations thereof.

6. The method of claim 5, wherein the antigen is an EBV, CMV or Adv antigen.

7. The method of claim 6, wherein the EBV, CMV and Adv antigens are encoded by pCK-IE1-IRES-pp65, pCK-E1dGALMP2-IRES-BZLF1 and pCK-HEXON-IRES-PENTON plasmid vectors, respectively as shown in FIGS. 8A, 8B and 8C, respectively.

8. The method of claim 1, wherein the antigen is a tumor antigen.

9. The method of claim 8, wherein the tumor antigen is selected from tumor specific proteins including MAGE1 (Melanoma Associated Antigen), AFP (Alpha Feto Protein) CEA (Carcinoembryonic Antige) or tyrosinase, tumor-specific antigens including telomerase, BCR-ABL (Abelson-Break point cluster) or RHAMM-R3 (receptor for hyaluronic acid-mediated motility), or WT1 (Wilms Tumor protein) and any mutant forms thereof.

10. The method of claim 9, wherein the tumor antigen is WT1.

11. The method of claim 10, wherein the WT1 is encoded by pCK-WT1 shown in FIG. 8D.

12. The method of claim 1, wherein the antigen-loaded activated B cells and the other mononuclear cells separated from the peripheral blood mononuclear cells at the first mixture or at the second mixture are present at a ratio of 1:

20 to 1;

1 in cell number.

13. The method of claim 1, wherein the human peripheral blood mononuclear cells are autologous or allogeneic.

14. A cell immunotherapeutic agent comprising the cells produced according to claim 1.