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One-Step Single-Chain Fv Recombinant Antibody-based Purification of gp96 for Vaccine Development¹

Institute for Cell Biology, Department of Immunology, University of Tübingen, D-72076 Tübingen, Germany [D. A-S., H-G. R., H. S.], and the Institute for Immunology, Department of Transplantation Immunology, University of Heidelberg, D-69120 Heidelberg, Germany [C. K., M. W., G. O., P. T.]

Heat shock proteins such as gp96 (grp94) isolated from tumor or infected cells are able to induce specific cytotoxic T-cell responses and protective immunity. To facilitate rapid and efficient isolation, we generated gp96-specific recombinant single-chain Fv (scFv) antibodies from a semisynthetic phage display library. When immobilized on Sepharose beads, these antibodies allow a high-yield, one-step purification of native gp96 molecules from both mouse and human tumor cell lysates. gp96 molecules eluted from these affinity columns under mild conditions are still capable of generating antigen-specific CTL responses in mice. Thus, scFv-purified gp96 is still associated with peptides; however, in contrast to conventionally purified gp96, scFv-isolated gp96 is free of contaminating material such as mitogenic concanavalin A and proteolytic cathepsins. With the help of these high-yield antibody columns, it is now possible to rapidly isolate immunogenic gp96-peptide complexes from small amounts of tumor material to a purity that allows their use in cancer immunotherapy protocols.