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 β 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] 1. Based on this finding, a chemically-modified synthetic peptide with the amino acid sequence corresponding to residues of β 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.