title: Generation of potent mouse monoclonal antibodies to self-proteins using T-cell epitope "tags".
abstract: 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.
authors.affiliation: a Department of Antibody Discovery and Protein Engineering; MedImmune Ltd. ; Abington , UK.
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Percival-Alwyn
authors.forename: Elizabeth
authors.initials: E
authors.lastname: England
authors.forename: Benjamin
authors.initials: B
authors.lastname: Kemp
authors.forename: Laura
authors.initials: L
authors.lastname: Rapley
authors.forename: Nicola H E
authors.initials: NH
authors.lastname: Davis
authors.forename: Grant R
authors.initials: GR
authors.lastname: McCarthy
authors.forename: Jayesh B
authors.initials: JB
authors.lastname: Majithiya
authors.forename: Dominic J
authors.initials: DJ
authors.lastname: Corkill
authors.forename: Sarah
authors.initials: S
authors.lastname: Welsted
authors.forename: Kevin
authors.initials: K
authors.lastname: Minton
authors.forename: E Suzanne
authors.initials: ES
authors.lastname: Cohen
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Robinson
authors.forename: Claire
authors.initials: C
authors.lastname: Dobson
authors.forename: Trevor C I
authors.initials: TC
authors.lastname: Wilkinson
authors.forename: Tristan J
authors.initials: TJ
authors.lastname: Vaughan
authors.forename: Maria A T
authors.initials: MA
authors.lastname: Groves
authors.forename: Natalie J
authors.initials: NJ
authors.lastname: Tigue
mesh_terms: D000818:Animals; D058846:Antibodies, Monoclonal, Murine-Derived / Q000276:immunology* / Q000494:pharmacology; D000918:Antibody Specificity; D001249:Asthma / Q000188:drug therapy / Q000276:immunology / Q000473:pathology; D002461:Cell Line, Transformed; D004167:Diphtheria Toxin / Q000737:chemistry / Q000276:immunology; D018984:Epitopes, T-Lymphocyte / Q000737:chemistry / Q000276:immunology*; D005260:Female; D006801:Humans; D000072179:Interleukin-1 Receptor-Like 1 Protein; D051379:Mice; D008807:Mice, Inbred BALB C; D051381:Rats; D018123:Receptors, Interleukin / Q000737:chemistry / Q000276:immunology*; D013744:Tetanus Toxin / Q000737:chemistry / Q000276:immunology
chemical_list: D058846:Antibodies, Monoclonal, Murine-Derived; D004167:Diphtheria Toxin; D018984:Epitopes, T-Lymphocyte; C486189:Il1rl1 protein, mouse; D000072179:Interleukin-1 Receptor-Like 1 Protein; D018123:Receptors, Interleukin; D013744:Tetanus Toxin
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