Method of making hybrid cells that express useful antibodies
This invention relates to a novel hybridoma strategy that uses CD27+ B cells cultured in vitro to induce IgM to IgG class switch prior to fusion with a fusion partner. Hybridomas resulting from the fusion between CD27+ B cells and a fusion partner cell line and antibodies secreted from the hybridomas are included in the invention.
1. A method of making a hybridoma, said method comprising
culturing peripheral blood mononuclear cells (PBMCs) enriched for CD27+ PBMCs in the presence of CD40L and cytokines comprising IL-4 and IL-10 for a period of time in vitro,
combining said cultured PBMCs enriched for CD27+ PBMCs with fusion partner cells, and
fusing said cultured PBMCs enriched for CD27+ PBMCs with said fusion partner cells, whereby fusion of CD27+ B cells of said cultured PBMCs with said fusion partner cells produces monoclonal antibody-producing hybridomas.
2. The method of claim 1 , wherein the concentration of IL-4 in the CD27+ enriched PBMC culture is about 2 ng/ml.
3. The method of claim 1 , wherein the concentration of IL-10 in the CD27+ enriched PBMC culture is about 10 ng/ml.
4. The method of claim 1 , wherein CD40L is provided in the form of CD40L displayed on the surface of tCD40L cells during the CD27+ enriched PBMC culturing.
5. The method of claim 1 , wherein the fusion partner cells ectopically expresses mIL-6 and hTERT.
6. The method of claim 1 , wherein said PBMSs enriched for CD27+ PBMCs are isolated from an immunized subject.
7. A method of producing a monoclonal antibody, the method comprising:
culturing peripheral blood mononuclear cells (PBMCs) enriched for CD27+ PBMCs in the presence of CD40L and cytokines comprising IL-4 and IL-10 for a period of time in vitro,
combining said cultured PBMCs enriched for CD27+ PBMCs with fusion partner cells,
fusing CD27+ B cells of said PBMCs enriched for CD27+ PBMCs with said fusion partner cells,
selecting a hybridoma that produces said monoclonal antibody, and
culturing said hybridoma to produce said monoclonal antibody.
8. The method of claim 7 , wherein the concentration of IL-4 in the CD27+ enriched PBMC culture is about 2 ng/ml.
9. The method of claim 7 , wherein the concentration of IL-10 in the CD27+ enriched PBMC culture is about 10 ng/ml.
10. The method of claim 7 , wherein CD40L is provided in the form of CD40L displayed on the surface of tCD40L cells during the CD27+ enriched PBMC culturing.
11. The method of claim 7 , wherein the fusion partner cells ectopically expresses mIL-6 and hTERT.
12. The method of claim 7 , wherein said enriched for CD27+ PBMCs are isolated from an immunized subject.
13. A method of making a library of hybridomas, said method comprising
culturing PBMCs enriched for CD27+ PBMCs in the presence CD40L and cytokines comprising IL-4 and IL-10 for a period of time in vitro,
combining said cultured PBMCs enriched for CD27+ PBMCs with fusion partner cells, and
fusing said cultured PBMCs enriched for CD27+ PBMCs with said fusion partner cells, whereby fusion of CD27+ B cells of said cultured PBMCs with said fusion partner cells produces a library of monoclonal antibody-producing hybridomas from said CD27+ B cells and said fusion partner cells.