This unit describes experimental procedures for development of therapeutic vaccines, particularly "second-generation" recombinant vaccines. Specifically, a general procedure for handling and culturing lymphoma cell lines in vitro and their subsequent challenge into syngeneic mice is described. Several protocols describe the production of various idiotype (Id)-based or cellular lymphoma vaccine formulations. In particular, the novel approach of rendering nonimmunogenic lymphoma-derived scFv or Id immunogenic by fusing it with a chemokine moiety is described. As an alternative, a protocol for expression and purification of these chemokine-fusion proteins from E. coli is included. A general procedure is included for cloning of cytokine genes, for example, murine GM-CSF, in A20 lymphoma cells. Support protocols are presented for chemically conjugating intact Ig protein with KLH to produce a prototype protein vaccine and using the Helios Gene Gun System to immunize mice with recombinant DNA tumor vaccines.
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