CHARACTERIZATION OF NATURALLY-OCCURRING AUTOANTIBODIES AGAINST TUMOR-NECROSIS-FACTOR-ALPHA (TNF-ALPHA) - IN-VITRO FUNCTION AND PRECISE EPITOPE MAPPING BY PHAGE EPITOPE LIBRARY

Naturally occurring autoantibodies against cytokines exist in the sera of patients with autoimmune diseases as well as in the sera of normal individuals. We report here that affinity-purified autoantibodies aga inst human TNF-alpha from one rheumatoid arthritis (RA) patient inhibi ted the cytotoxic effect of TNF-alpha on the mouse fibrosarcoma cell l ine WEHI 164, by 50%. In an attempt to predict the autoantibodies' rec ognition site on TNF-alpha protein we screened a random nanopeptide ph age library with the affinity-purified TNF-alpha autoantibodies. Among 63 random selected clones, 46 clones carried the sequence ASSLLASSP, NSSPYLNTK or PQSPGSSFP. Frequency analysis of the relative occurrence of the 20 amino acids in the nanopeptides displayed by 50 random bacte riophages picked before selection and 63 after selection to bind to TN F-alpha autoantibodies indicated that proline (P < 0.0003) and serine (P < 0.04) are involved in the binding of the autoantibodies to the ph ages. Furthermore, we demonstrated that three synthetic peptides (ASSL LASSP, NSSPYLNTK and PPLKPVIDE) displayed by the selected phages reduc ed the binding of the autoantibodies to TNF-alpha protein by 50%. Inte restingly, the sera of mice (BALB/c) immunized with phages displaying ASSLLASSP and NSSPYLNTK peptide showed an anti-TNF-alpha response as d etected by ELISA. This response was not found in mice immunized with t he wild type phage. Thus, the recombinant phages selected from the pha ge libraries could be used as carrier for immunization, and therefore as a tool for vaccine development. This work sets the stage for experi ments designed to isolate ligands for protective antibodies.