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 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.
Arnold-Schild
D
D
Institute for Cell Biology, Department of Immunology, University of Tübingen, Germany.
Kleist
C
C
Welschof
M
M
Opelz
G
G
Rammensee
H G
HG
Schild
H
H
Terness
P
P
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D000951:Antigens, Neoplasm / Q000276:immunology* / Q000302:isolation & purification*; D000954:Antigens, Surface / Q000276:immunology / Q000302:isolation & purification; D019496:Cancer Vaccines / Q000276:immunology / Q000302:isolation & purification*; D002847:Chromatography, Agarose / Q000379:methods; D006360:Heat-Shock Proteins / Q000276:immunology / Q000302:isolation & purification; D006801:Humans; D007128:Immunoglobulin Fragments / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D008213:Lymphocyte Activation / Q000276:immunology; D051379:Mice; D008810:Mice, Inbred C57BL; D008815:Mice, Inbred Strains; D008969:Molecular Sequence Data; D019151:Peptide Library; D011233:Precipitin Tests; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D017386:Sequence Homology, Amino Acid; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology; D014407:Tumor Cells, Cultured / Q000276:immunology
D000951:Antigens, Neoplasm; D000954:Antigens, Surface; D019496:Cancer Vaccines; D006360:Heat-Shock Proteins; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D011994:Recombinant Proteins; C052646:sarcoma glycoprotein gp96 rejection antigens