One-step single-chain Fv recombinant antibody-based purification of gp96 for vaccine development

Heat shock proteins such as gp96 (grp94) isolated from tumor or infected ce lls are able to induce specific cytotoxic T-cell responses and protective i mmunity. To facilitate rapid and efficient isolation, we generated gp96-spe cific recombinant single-chain Fv (scFv) antibodies from a semisynthetic ph age display library. When immobilized on Sepharose beads, these antibodies allow a high-yield, one-step purification of native gp96 molecules from bot h mouse and human tumor cell lysates. gp96 molecules eluted from these affi nity columns under mild conditions are still capable of generating antigen- specific CTL responses in mice. Thus, scFv-purified gp96 is still associate d with peptides; however, in contrast to conventionally purified gp96, scFv -isolated gp96 is free of contaminating material such as mitogenic concanav alin A and proteolytic cathepsins, With the help of these high-yield antibo dy columns, it is now possible to rapidly Isolate immunogenic gp96-peptide complexes from small amounts of tumor material to a purity that allows thei r use in cancer immunotherapy protocols.