Methods for in vitro memory b cell differentiation and transduction with vsv-g pseudotyped viral vectors

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.

What is claimed is: 
     
         1 . A method for expressing a nucleic acid of interest in a B cell comprising, (a) contacting memory B cells with a composition comprising CD40L in combination with a B cell activating factor comprising one or more of the factors selected from the group consisting of IL-2, IL-10, IL-15, and p-ODN;
 (b) contacting the B cells of (a) with a B cell activating factor comprising one or more of the factors selected from the group consisting of 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 in (b) 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 a B cell activating factor comprising one or more of the factors selected from the group consisting of 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 transduction efficiency is about 20%. 
     
     
         4 . The method of  claim 1 , wherein the transduction efficiency is higher than 20%. 
     
     
         5 . The method of  claim 1 , wherein the retroviral vector is a lentiviral vector. 
     
     
         6 . The method of  claim 1  wherein the nucleic acid of interest comprises a nucleic acid encoding at least an immunoglobulin VL and VH region. 
     
     
         7 . The method of  claim 1  wherein the nucleic acid of interest encodes an antibody protein, or antigen-binding fragment thereof, a fusion protein or a DNA-encoded small molecule. 
     
     
         8 . The method of  claim 7  wherein the antibody is an anti-HIV antibody, an anti-RNA antibody, or an antibody that binds a protein involved in immune regulation. 
     
     
         9 . The method of  claim 1  wherein the memory B cells are isolated from peripheral blood. 
     
     
         10 . The method of  claim 1  wherein the B cells of (b) are CD20−, CD38+ and CD138−. 
     
     
         11 . The method of  claim 1  wherein the B cells of (c) are CD20−, CD38+ and CD138+.