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US 11,352,603 B2Granted
Methods for in vitro memory B cell differentiation and transduction with VSV-G pseudotyped viral vectors
AssigneeImmusoft Corporation
InventorsMei Xu, Matthew Rein Scholz, Eric J. Herbig
Life of this patent
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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.
Claims18
An in vitro culture method for expressing a nucleic acid of interest in B cells comprising,
- (a) contacting B cells that are CD20+,CD38−, and CD138− with a composition comprising multimerized soluble CD40L in combination with IL-2, IL-10, IL-15, and CpG oligodeoxynucleotides (p-ODN) until the B cells become CD20−, CD38−, CD138−, and CD27+;
- (b) removing the composition comprising multimerized soluble CD40L in combination with IL-2, IL-10, IL-15, and CpG oligodeoxynucleotides (p-ODN) from the B cells and contacting the remaining B cells with IL-2, IL-10, IL-6 and IL-15 until the B cells are mostly CD20−, CD38+, and CD138−;
- and
- (c) transducing the B cells after step (b) on day 5 after the start of in vitro culture with a retroviral vector pseudotyped with VSV-G, wherein said retroviral vector comprises the nucleic acid of interest operably linked to a promoter, wherein the transduction efficiency is about 20%, thereby expressing the nucleic acid of interest in B cells.
1 more element
An in vitro culture method for expressing a nucleic acid of interest in B cells comprising,
- (a) contacting a B cell population comprising memory B cells with a composition comprising multimerized soluble CD40L in combination with IL-2, IL-10, IL-15, and CpG oligodeoxynucleotides (p-ODN) for about 3 days;
- (b) removing the composition comprising multimerized soluble CD40L in combination with IL-2, IL-10, IL-15, and CpG oligodeoxynucleotides (p-ODN) from the B cells and contacting the remaining B cells with IL-2, IL-10, IL-6 and IL-15, under conditions such that the B cells express CD38;
- and
- (c) transducing the B cells after step (b) on day 5 after the start of in vitro culture with a retroviral vector pseudotyped with VSV-G, wherein said retroviral vector comprises the nucleic acid of interest operably linked to a promoter,
- thereby expressing the nucleic acid of interest in B cells, wherein transduction efficiency is about 20%.
2 more elements
Dependencies are inferred from the claim text.