title: Anti-melanoma antibodies from melanoma patients immunized with genetically modified autologous tumor cells: selection of specific antibodies from single-chain Fv fusion phage libraries.
abstract: Fusion phage libraries expressing single-chain Fv antibodies were constructed from the peripheral blood lymphocytes of two melanoma patients who had been immunized with autologous melanoma cells transduced the gamma-interferon gene to enhance immunogenicity, in a trial conducted at another institution. Anti-melanoma antibodies were selected from each library by panning the phage against live cultures of the autologous tumor. After two or three rounds of panning, clones of the phage were tested by ELISA for binding to the autologous tumor cells; > 90% of the clones tested showed a strong ELISA reaction, demonstrating the effectiveness of the panning procedure for selecting antimelanoma antibodies. The panned phage population was extensively absorbed against normal melanocytes to enrich for antibodies that react with melanoma cells but not with melanocytes. The unabsorbed phage were cloned, and the specificities of the expressed antibodies were individually tested by ELISA with a panel of cultured human cells. The first tests were done with normal endothelial and fibroblast cells to identify antibodies that do not react, or react weakly, with two normal cell types, indicating some degree of specificity for melanoma cells. The proportion of phage clones expressing such antibodies was approximately 1%. Those phage were further tested by ELISA with melanocytes, several melanoma lines, and eight other tumor lines, including a glioma line derived from glial cells that share a common lineage with melanocytes. The ELISA tests identified three classes of anti-melanoma antibodies, as follows: (i) a melanoma-specific class that reacts almost exclusively with the melanoma lines; (ii) a tumor-specific class that reacts with melanoma and other tumor lines but does not react with the normal melanocyte, endothelial and fibroblast cells; and (iii) a lineage-specific class that reacts with the melanoma lines, melanocytes, and the glioma line but does not react with the other lines. These are rare classes from the immunized patients' repertoires of anti-melanoma antibodies, most of which are relatively nonspecific anti-self antibodies. The melanoma-specific class was isolated from one patient, and the lineage-specific class was isolated from the other patient, indicating that different patients can have markedly different responses to the same immunization protocol. The procedures described here can be used to screen the antibody repertoire of any person with cancer, providing access to an enormous untapped pool of human monoclonal anti-tumor antibodies with clinical and research potential.
authors.affiliation: Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA.
authors.forename: X
authors.initials: X
authors.lastname: Cai
authors.forename: A
authors.initials: A
authors.lastname: Garen
mesh_terms: D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000276:immunology*; D001323:Autoantibodies / Q000096:biosynthesis / Q000097:blood*; D001435:Bacteriophages / Q000235:genetics; D001483:Base Sequence; D002478:Cells, Cultured; D017931:DNA Primers; D004730:Endothelium, Vascular / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D015723:Gene Library; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000276:immunology; D007133:Immunoglobulin Joining Region / Q000276:immunology; D007147:Immunoglobulin Light Chains / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000276:immunology; D007371:Interferon-gamma / Q000096:biosynthesis; D008214:Lymphocytes / Q000276:immunology; D008545:Melanoma / Q000097:blood / Q000276:immunology*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D014407:Tumor Cells, Cultured; D014471:Umbilical Veins
chemical_list: D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D017931:DNA Primers; D007143:Immunoglobulin Heavy Chains; D007133:Immunoglobulin Joining Region; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D007371:Interferon-gamma