title: Application of murine T-T hybridoma cells to in vitro potency assay of human synthetic peptide vaccines.
abstract: Recently, it has been shown that the immunization of mice with an 18 amino acid synthetic peptide corresponding to the third hypervariable region of MHC class II beta chain can induce a specific antibody response against MHC class II molecules, and can be utilized in the prevention and treatment of experimental allergic encephalomyelitis (EAE) [Proc. Natl. Acad. Sci. 1994, 91, 8005-8009]. Based on this finding, a chemically-modified synthetic peptide with the amino acid sequence corresponding to residues of beta 57-76 from human HLA-DR4Dw4 (DR4/1 peptide) is being clinically investigated for the treatment of rheumatoid arthritis in human. The present study describes the development of a novel in vitro potency assay for human HLA-DR4/1 peptide using cloned murine T-T hybridoma cells. Several mouse strains were immunized with the DR4/1 peptide and their lymph node T cell proliferation was measured in the presence of syngeneic APCs and the DR4/1 peptide. T cells isolated from the peptide primed-B10. PL mouse strain, which showed the highest recall response in this assay, were fused with BW5147 lymphoma cells to generate DR4/1 peptide-specific T-T hybridoma clones. Cloned hybridoma cells were characterized for peptide specificity and MHC class II restriction, and used to monitor the biological activity of various DR4/1 peptide preparations. The potency of peptide batches were assessed by measuring the IL-2 secretion of cloned T-T hybridoma cells upon TCR engagement in an antigen-specific manner. The quantitative detection of IL-2 was performed by measuring [3H]thymidine incorporation of HT-2 cells or directly by ELISA. These results demonstrate that peptide-specific murine T-T hybridoma clones can be successfully utilized to monitor biological activity of synthetic peptides by measuring T cell-mediated immunological responses. Development of such in vitro potency assay for synthetic peptides may have broad applications for vaccines related to immunological disorders.
authors.affiliation: Anergen Inc., Redwood City, CA 94063, USA.
authors.forename: S C
authors.initials: SC
authors.lastname: Yu
authors.forename: B
authors.initials: B
authors.lastname: Nag
mesh_terms: D000818:Animals; D017252:CD3 Complex / Q000276:immunology; D015704:CD4 Antigens / Q000276:immunology; D002455:Cell Division / Q000276:immunology; D002462:Cell Membrane / Q000276:immunology; D005260:Female; D000949:Histocompatibility Antigens Class II / Q000276:immunology; D006801:Humans; D006825:Hybridomas; D007111:Immunity, Cellular; D007376:Interleukin-2 / Q000096:biosynthesis; D051379:Mice; D011948:Receptors, Antigen, T-Cell / Q000276:immunology; D015203:Reproducibility of Results; D013601:T-Lymphocytes / Q000166:cytology / Q000276:immunology*; D014614:Vaccines, Synthetic / Q000276:immunology*
chemical_list: D017252:CD3 Complex; D015704:CD4 Antigens; D000949:Histocompatibility Antigens Class II; D007376:Interleukin-2; D011948:Receptors, Antigen, T-Cell; D014614:Vaccines, Synthetic