Immunization of mice or rats with a "non-self" protein is a commonly used method to obtain monoclonal antibodies, and relies on the immune system's ability to recognize the immunogen as foreign. Immunization of an antigen with 100% identity to the endogenous protein, however, will not elicit a robust immune response. To develop antibodies to mouse proteins, we focused on the potential for breaking such immune tolerance by genetically fusing two independent T-cell epitope-containing sequences (from tetanus toxin (TT) and diphtheria toxin fragment A (DTA)) to a mouse protein, mouse ST2 (mST2). Wild-type CD1 mice were immunized with three mST2 tagged proteins (Fc, TT and DTA) and the specific serum response was determined. Only in mice immunized with the T-cell epitope-containing antigens were specific mST2 serum responses detected; hybridomas generated from these mice secreted highly sequence-diverse IgGs that were capable of binding mST2 and inhibiting the interaction of mST2 with its ligand, mouse interleukin (IL)-33 (mIL-33). Of the hundreds of antibodies profiled, we identified five potent antibodies that were able to inhibit IL-33 induced IL-6 release in a mast cell assay; notably one such antibody was sufficiently potent to suppress IL-5 release and eosinophilia infiltration in an Alternaria alternata challenge mouse model of asthma. This study demonstrated, for the first time, that T-cell epitope-containing tags have the ability to break tolerance in wild-type mice to 100% conserved proteins, and it provides a compelling argument for the broader use of this approach to generate antibodies against any mouse protein or conserved ortholog.
Keywords: APC, antigen presenting cells; Antibody generation; CDR, complementarity determining region; CHO, Chinese hamster ovary; DMEM, dulbecco's modified eagles' medium; DTA, diphtheria toxin; ELISA, enzyme-linked immunosorbent assay; HLA, human leukocyte antigen; HTRF, homogenous time-resolved fluorescence; IL, interleukin; ILC2, type 2 innate lymphoid cells; IgG, immunoglobulin G; MHC, major histocompatibility complex; PADRE, pan HLA-DR-binding T cell epitope; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel; SLE, systemic lupus erythematosus; T-cell epitopes; TCR, T cell receptor; TT, tetanus tosxin; VH, variable region of immunoglobulin heavy chain; VL, variable region of immunoglobulin light chain; diphtheria toxin; hybridoma; immunological tolerance; mST2, mouse ST2; mouse ST2; tetanus toxin.