Human monoclonal autoantibody fragments from combinatorial antibody libraries directed to the U1snRNP associated U1C protein; epitope mapping, immunolocalization and V-gene usage

To study the localization and function of the U1snRNP associated U1C protei n, so far only human sera From systemic lupus erythematosus (SLE) overlap s yndrome patients have been used. Here we report for the first time the isol ation of human monoclonal anti-U1C autoantibody fragments from IgG derived combinatorial and semi-synthetic human antibody libraries. Two classes of h uman monoclonal anti-U1C (auto)antibodies were found: specific anti-U1C aut oantibodies, recognizing U1C only, and crossreactive antibodies which also react with U1A and Sm-B/B' proteins. The heavy chains (V-H genes) of all fi ve antibodies from the semi-synthetic libraries and two of the three U1C-sp ecific patient derived autoantibody fragments are encoded by V(H)3 genes, i n which V-H 3-30 (DP-49) was overrepresented. The heavy chain of the two cr oss-reactive autoantibodies are derived from the 3-07 (DP-54) gene. Three e pitope regions on the U1C protein are targeted by these antibodies. (1) Fou r U1C specific antibodies recognize an N-terminal region of U1C in which am ino acids 30-63 are essential for recognition, (2) two antibodies recognize only the complete U1C protein, and (3) two cross-reactive and one U1C spec ific antibody recognize the C-terminal domain in which amino acids 98-126 a re critical for recognition. The two cross-reactive antibodies (K11 and K15 ) recognize the proline-rich region of the U1C protein (amino acids 98-126) and cross-react with proline-rich regions in Sm-B/B' (amino acids 163-184) and U1A (amino acids 187-204). All 10 antibody fragments are able to immun oprecipitate the native U1snRNP particle. The two cross-reactive antibodies immunoprecipitate the other Sm containing snRNPs as well. Using confocal i mmunofluorescence microscopy we could show that the major part of the U1C p rotein is localized within the coiled body structure. (C) 1999 Elsevier Sci ence Ltd. All rights reserved.