Methods for in vitro memory B cell differentiation and transduction with VSV-G pseudotyped viral vectors

• US 10,240,125 B2
• Filed: 03/14/2014
• Issued: 03/26/2019
• Est. Priority Date: 03/14/2013
• Status: Active Grant

• Current Assignee: Immusoft Corporation
• Original Assignee: Immusoft Corporation
• Inventors: Xu, Mei, Scholz, Matthew Rein, Herbig, Eric J.
• Primary Examiner(s): Nguyen, Quang
• Application Number:
  US14/776,283
• Publication Number:
  US 20160046908A1
• Time in Patent Office: 1,838 Days
• CPC Class Codes: C12N 2501/2302 Interleukin-2 (IL-2)
• CPC Class Codes: C12N 2501/2306 Interleukin-6 (IL-6)
• CPC Class Codes: C12N 2501/231 Interleukin-10 (IL-10)
• CPC Class Codes: C12N 2501/2315 Interleukin-15 (IL-15)
• CPC Class Codes: C12N 2501/24 Interferons [IFN]
• CPC Class Codes: C12N 2501/52 CD40, CD40-ligand (CD154)
• CPC Class Codes: C12N 2510/02 Cells for production
• CPC Class Codes: C12N 5/0635 B lymphocytes

Abstract

The present disclosure relates to the in vitro differentiation of memory B cells to plasmablasts or plasma cells and genetic modification of these cells to express a protein of interest, such as a specific antibody or other protein therapeutic.

Claims

1. An in vitro culture method for expressing a nucleic acid of interest in a B cell comprising,

(a) contacting memory B cells with a composition comprising multimerized soluble CD40L in combination with IL-2, IL-10, IL15 and class P CpG oligodeoxynucleotides (p-ODN) (“

CD40L composition”

) for about 3 days;

(b) removing the CD40L composition from the B cells of (a) and contacting the B cells of (a) with IL-2, IL-10, IL-6 and IL-15 under conditions such that the B cells in (b) express CD38;

(c) transducing the B cells of (b) on day 5 of the in vitro cell culture with a retroviral vector pseudotyped with VSV-G, wherein said retroviral vector comprises the nucleic acid of interest operably linked to a promoter; and

(d) contacting the transduced B cells of (c) with IFN-α

, IFN-δ

, IL-6 and IL-15;

thereby expressing the nucleic acid of interest in the B cell.

2. The method of claim 1, wherein the CD40L is sCD40L-his.

3. The method of claim 1, wherein the retroviral vector is a lentiviral vector.

4. The method of claim 1, wherein the nucleic acid of interest comprises a nucleic acid encoding at least an immunoglobulin VL and VH region.

5. The method of claim 1, wherein the nucleic acid of interest encodes an antibody protein or an antigen-binding fragment thereof, a fusion protein, or a DNA-encoded small molecule.

6. The method of claim 5, wherein the antibody is an anti-HIV antibody, an anti-RNA antibody, or an antibody that binds a protein involved in immune regulation.

7. The method of claim 1, wherein the memory B cells are isolated from peripheral blood.

8. The method of claim 1, wherein the B cells of (b) are CD20−

, CD38+and CD138−

.

9. The method of claim 1, wherein the B cells of (c) are CD20−

, CD38+and CD138+.

10. The method of claim 1, wherein the nucleic acid of interest encodes a cell surface receptor, a secreted protein, or an adhesion molecule.

11. The method of claim 10, wherein the secreted protein is a cytokine or chemokine.

12. The method of claim 1, wherein the nucleic acid of interest encodes an immunologically active protein.

13. The method of claim 1, wherein the B cell is a human B cell.

14. The method of claim 1, wherein the method is carried out in the absence of feeder cells.